RESUMO
OBJECTIVES: We aimed to determine the presence, amount and origin of microchimerism in peripheral blood of pregnant and non-pregnant parous women with systemic lupus erythematosus (SLE) as compared to control subjects. METHODS: We performed a comparative study in which peripheral blood was drawn from eleven female non-pregnant SLE-patients and 22 control subjects, and from six pregnant SLE-patients and eleven control subjects during gestation and up to six months postpartum. Quantitative PCR for insertion-deletion polymorphisms and null alleles was used to detect microchimerism in peripheral blood mononuclear cells and granulocytes. RESULTS: Microchimerism was detected more often in non-pregnant SLE-patients than control subjects (54.4% vs. 13.6%, respectively; p=0.03). When present, the median total number of foetal chimeric cells was 5 gEq/106 in patients and 2.5gEq/106 in control subjects (p=0.048). Microchimerism was mostly foetal in origin; maternal microchimerism was detected in one patient and one control subject. In control subjects, microchimerism was always derived from only one source whereas in 50% of patients it originated from multiple sources. The pregnant patients had a significantly higher median number of foetal chimeric cells in the granulocyte fraction just after delivery than control subjects (7.5 gEq/106 vs. 0 gEq/106, respectively; p=0.02). CONCLUSIONS: Just after delivery, SLE-patients had more microchimerism than control subjects. Three months post-partum, microchimerism was no longer detectable, only to reappear many years after the last pregnancy, more often and at higher levels in SLE-patients than in control subjects. This suggests that these chimeric cells may originate from non-circulating foetal chimeric stem cells.
Assuntos
Lúpus Eritematoso Sistêmico , Complicações na Gravidez , Gravidez , Humanos , Feminino , Quimerismo , Leucócitos Mononucleares , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: An excessive release and impaired degradation of neutrophil extracellular traps (NETs) leads to the continuous exposure of NETs to the endothelium in a variety of hematologic and autoimmune disorders, including lupus nephritis. This study aims to unravel the mechanisms through which NETs jeopardize vascular integrity. APPROACH AND RESULTS: Microvascular and macrovascular endothelial cells were exposed to NETs, and subsequent effects on endothelial integrity and function were determined in vitro and in vivo. We found that endothelial cells have a limited capacity to internalize NETs via the receptor for advanced glycation endproducts. An overflow of the phagocytic capacity of endothelial cells for NETs resulted in the persistent extracellular presence of NETs, which rapidly altered endothelial cell-cell contacts and induced vascular leakage and transendothelial albumin passage through elastase-mediated proteolysis of the intercellular junction protein VE-cadherin. Furthermore, NET-associated elastase promoted the nuclear translocation of junctional ß-catenin and induced endothelial-to-mesenchymal transition in cultured endothelial cells. In vivo, NETs could be identified in kidney samples of diseased MRL/lpr mice and patients with lupus nephritis, in whom the glomerular presence of NETs correlated with the severity of proteinuria and with glomerular endothelial-to-mesenchymal transition. CONCLUSIONS: These results indicate that an excess of NETs exceeds the phagocytic capacity of endothelial cells for NETs and promotes vascular leakage and endothelial-to-mesenchymal transition through the degradation of VE-cadherin and the subsequent activation of ß-catenin signaling. Our data designate NET-associated elastase as a potential therapeutic target in the prevention of endothelial alterations in diseases characterized by aberrant NET release.
Assuntos
Transição Epitelial-Mesenquimal , Armadilhas Extracelulares/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Glomérulos Renais/metabolismo , Nefrite Lúpica/metabolismo , Neutrófilos/metabolismo , Adulto , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar , Clatrina/metabolismo , Modelos Animais de Doenças , Endocitose , Armadilhas Extracelulares/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Elastase de Leucócito/metabolismo , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Camundongos Endogâmicos CBA , Camundongos Endogâmicos MRL lpr , Neutrófilos/imunologia , Neutrófilos/patologia , Fagocitose , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Fatores de Tempo , Adulto Jovem , beta Catenina/metabolismoRESUMO
Vitamin D plays an important role in renal (patho)physiology. Patients with glomerular diseases have an injured renal filtration barrier, leading to proteinuria and reduced renal function. An impaired renal function also leads to 1,25-vitamin D3 deficiency as a result of reduced renal 1α-hydroxylase activity. Vitamin D treatment to reduce proteinuria remains controversial, although there is an inverse correlation between vitamin D levels and proteinuria. Herein, we showed that 1,25-vitamin D3-deficient 25-hydroxy-vitamin-D3-1α-hydroxylase knockout mice and 1,25-vitamin D3-deficient rats develop podocyte injury and renal dysfunction. Glomerular injury was characterized by proteinuria and partial podocyte foot process effacement. Expression of nephrin, podocin, desmin, and transient receptor potential channel C6 in the podocyte was significantly altered in 1,25-vitamin D3-deficient animals. Supplementation with 1,25-vitamin D3 or 1,25-vitamin D2 prevented podocyte effacement or reversed glomerular and tubulointerstitial damage in 1,25-vitamin D3-deficient animals, thereby preserving and restoring renal function, respectively. The effect of 1,25-vitamin D3 deficiency and 1,25-vitamin D3 and 1,25-vitamin D2 repletion on proteinuria could not be explained by hypocalcemia, changes in parathyroid hormone, or fibroblast growth factor 23. This study demonstrates that 1,25-vitamin D3 deficiency directly leads to renal injury in rodents. Translated to human subjects, this would underline the need for early vitamin D supplementation in patients with glomerular disease and chronic renal insufficiency, which might inhibit or potentially reverse renal injury.
Assuntos
Albuminúria/etiologia , Albuminúria/metabolismo , Colecalciferol/deficiência , Nefropatias/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Animais , Glomérulos Renais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hormônio Paratireóideo/metabolismo , Ratos , Ratos WistarRESUMO
Heparanase, a heparan sulfate (HS)--specific endoglucuronidase, mediates the onset of proteinuria and renal damage during experimental diabetic nephropathy. Glomerular heparanase expression is increased in most proteinuric diseases. Herein, we evaluated the role of heparanase in two models of experimental glomerulonephritis, being anti-glomerular basement membrane and lipopolysaccharide-induced glomerulonephritis, in wild-type and heparanase-deficient mice. Induction of experimental glomerulonephritis led to an increased heparanase expression in wild-type mice, which was associated with a decreased glomerular expression of a highly sulfated HS domain, and albuminuria. Albuminuria was reduced in the heparanase-deficient mice in both models of experimental glomerulonephritis, which was accompanied by a better renal function and less renal damage. Notably, glomerular HS expression was preserved in the heparanase-deficient mice. Glomerular leukocyte and macrophage influx was reduced in the heparanase-deficient mice, which was accompanied by a reduced expression of both types 1 and 2 helper T-cell cytokines. In vitro, tumor necrosis factor-α and lipopolysaccharide directly induced heparanase expression and increased transendothelial albumin passage. Our study shows that heparanase contributes to proteinuria and renal damage in experimental glomerulonephritis by decreasing glomerular HS expression, enhancing renal leukocyte and macrophage influx, and affecting the local cytokine milieu.
Assuntos
Nefropatias Diabéticas/metabolismo , Membrana Basal Glomerular/metabolismo , Glomerulonefrite/etiologia , Glomerulonefrite/metabolismo , Glucuronidase/metabolismo , Doença Aguda , Animais , Heparitina Sulfato/metabolismo , Camundongos Endogâmicos C57BL , Proteinúria/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Diabetic nephropathy (DN) is the leading cause of CKD in the Western world. Endothelin receptor antagonists have emerged as a novel treatment for DN, but the mechanisms underlying the protective effect remain unknown. We previously showed that both heparanase and endothelin-1 are essential for the development of DN. Here, we further investigated the role of these proteins in DN, and demonstrated that endothelin-1 activates podocytes to release heparanase. Furthermore, conditioned podocyte culture medium increased glomerular transendothelial albumin passage in a heparanase-dependent manner. In mice, podocyte-specific knockout of the endothelin receptor prevented the diabetes-induced increase in glomerular heparanase expression, consequent reduction in heparan sulfate expression and endothelial glycocalyx thickness, and development of proteinuria observed in wild-type counterparts. Our data suggest that in diabetes, endothelin-1 signaling, as occurs in endothelial activation, induces heparanase expression in the podocyte, damage to the glycocalyx, proteinuria, and renal failure. Thus, prevention of these effects may constitute the mechanism of action of endothelin receptor blockers in DN.
Assuntos
Endotelina-1/fisiologia , Glucuronidase/fisiologia , Glicocálix/enzimologia , Glomérulos Renais/enzimologia , Glomérulos Renais/ultraestrutura , Proteinúria/etiologia , Animais , Nefropatias Diabéticas/etiologia , Masculino , Camundongos , Podócitos/enzimologiaRESUMO
Proteinuria is one of the first clinical signs of diabetic nephropathy and an independent predictor for the progression to renal failure. Cathepsin L, a lysosomal cysteine protease, can be involved in the development of proteinuria by degradation of proteins that are important for normal podocyte architecture, such as the CD2-associated protein, synaptopodin, and dynamin. Cathepsin L also activates heparanase, a heparan sulfate endoglycosidase previously shown to be crucial for the development of diabetic nephropathy. Here, we evaluated the exact mode of action of cathepsin L in the development of proteinuria in streptozotocin-induced diabetes. Cathepsin L-deficient mice, in contrast to their wild-type littermates, failed to develop albuminuria, mesangial matrix expansion, tubulointerstitial fibrosis, and renal macrophage influx and showed a normal renal function. In wild-type mice the early development of albuminuria correlated with the activation of heparanase and loss of heparan sulfate expression, whereas loss of synaptopodin expression and podocyte damage occurred at a later stage. Thus, cathepsin L is causally involved in the pathogenesis of experimental diabetic nephropathy. Most likely, cathepsin L-dependent heparanase activation is crucial for the development of albuminuria and renal damage.
Assuntos
Catepsina L/metabolismo , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/etiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Dinaminas/metabolismo , Glucuronidase/metabolismo , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismoRESUMO
The glomerular filtration barrier consists of podocytes, the glomerular basement membrane, and endothelial cells covered with a glycocalyx. Heparan sulphate (HS) in the glomerular filtration barrier is reduced in patients with proteinuria, which is associated with increased expression of the HS-degrading enzyme heparanase. Previously, we showed that heparanase is essential for the development of proteinuria in experimental diabetic nephropathy. Vitamin D supplementation reduces podocyte loss and proteinuria in vitro and in vivo. Therefore, we hypothesize that vitamin D reduces proteinuria by reducing glomerular heparanase. Adriamycin-exposed rats developed proteinuria and showed increased heparanase expression, which was reduced by 1,25-dihydroxyvitamin D3 (1,25-D3) treatment. In vitro, adriamycin increased heparanase mRNA in the podocyte, which could be corrected by 1,25-D3 treatment. In addition, 1,25-D3 treatment reduced transendothelial albumin passage after adriamycin stimulation. In line with these results, we showed direct binding of the vitamin D receptor to the heparanase promoter, and 1,25-D3 dose-dependently reduced heparanase promoter activity. Finally, 1,25-D3-deficient 25-hydroxy-1α-hydroxylase knockout mice developed proteinuria and showed increased heparanase, which was normalized by 1,25-D3 treatment. Our data suggest that the protective effect of vitamin D on the development of proteinuria is mediated by inhibiting heparanase expression in the podocyte.
Assuntos
Calcitriol/farmacologia , Glucuronidase/metabolismo , Podócitos/enzimologia , Proteinúria/metabolismo , Animais , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Heparitina Sulfato/metabolismo , Camundongos , Camundongos Knockout , Podócitos/efeitos dos fármacos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Slit diaphragm and podocyte damage is crucial in the pathogenesis of proteinuria in diabetic nephropathy (DNP). Gain-of-function mutations in TRPC6, a slit diaphragm-associated ion channel, cause glomerulosclerosis; TRPC6 expression is increased in acquired glomerular disease. Hyperglycemia and high intrarenal angiotensin II (AngII) levels could contribute to podocyte injury in DNP. We determined whether glucose regulates TRPC6 expression and TRPC6-mediated Ca(2+) influx into the podocyte and whether these effects are AngII dependent. High glucose levels increased TRPC6 mRNA and protein expression in cultured podocytes; however, TRPC1 and TRPC5 mRNA expression was unaltered. AngII and inducing podocyte injury also specifically increased TRPC6 expression. Angiotensin receptor blockade and inhibition of local AngII production through angiotensin-converting enzyme inhibition prevented glucose-mediated increased TRPC6 expression. In addition, high glucose concentration pretreatment enhanced Ca(2+) influx in podocytes, which was prevented by concomitant angiotensin receptor blockade application and TRPC6 knockdown. Studies with a TRPC6 luciferase promoter construct demonstrated a glucose concentration-dependent effect on TRPC6 promoter activity. In vivo, podocyte TRPC6 protein expression was increased in proteinuric streptozotocin-induced diabetic rats. These data suggest that glucose can activate a local renin-angiotensin system in the podocyte, leading to increased TRPC6 expression, which enhances TRPC6-mediated Ca(2+) influx. Regulation of TRPC6 expression could be an important factor in podocyte injury due to chronic hyperglycemia and the antiproteinuric effect of angiotensin receptor blockade or angiotensin-converting enzyme inhibition in DNP.
Assuntos
Angiotensina II/metabolismo , Nefropatias Diabéticas/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Podócitos/metabolismo , Canais de Cátion TRPC/biossíntese , Animais , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Camundongos , Camundongos Knockout , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Podócitos/patologia , Ratos , Ratos Wistar , Sistema Renina-Angiotensina/genética , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6RESUMO
Mycophenolic acid (MPA) has become the cornerstone in the treatment of lupus nephritis. However, response rates are still far from ideal in clinical trials. Uncertainty exists regarding the correct dosing of MPA, and the recommended doses vary between recently published guidelines. Side effects are an additional problem resulting in frequent dose reduction and possible suboptimal exposure.In this review, we discuss the large variability between patients in drug exposure to MPA and the evidence for a relationship between drug exposure and efficacy in lupus nephritis. Methods for drug monitoring of MPA are discussed, and based on the current literature, we suggest as potential target levels a pre-dose level of 3.0 mg/L and an area under the concentration-versus-time curve between 35 and 45 mg h/L.Therapeutic drug monitoring may improve response rates in lupus nephritis by preventing low exposure and at the same time may reduce unnecessary side effects in patients who have high drug exposure with standard dose MPA. We specifically advise assessment of MPA drug exposure early after start of treatment and before concluding that treatment with MPA has failed.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Monitoramento de Medicamentos/estatística & dados numéricos , Nefrite Lúpica/tratamento farmacológico , Ácido Micofenólico/uso terapêutico , Humanos , Nefrite Lúpica/diagnósticoRESUMO
The glomerular endothelial glycocalyx is postulated to be an important modulator of permeability and inflammation. The glycocalyx consists of complex polysaccharides, the main functional constituent of which, heparan sulfate (HS), is synthesized and modified by multiple enzymes. The N-deacetylase-N-sulfotransferase (Ndst) enzymes initiate and dictate the modification process. Here we evaluated the effects of modulation of HS in the endothelial glycocalyx on albuminuria and glomerular leukocyte influx using mice deficient in endothelial and leukocyte Ndst1 (TEKCre+/Ndst1flox/flox). In these mice, glomerular expression of a specific HS domain was significantly decreased, whereas the expression of other HS domains was normal. In the endothelial glycocalyx, this specific HS structure was not associated with albuminuria or with changes in renal function. However, glomerular leukocyte influx was significantly reduced during antiglomerular basement membrane nephritis, which was associated with less glomerular injury and better renal function. In vitro decreased adhesion of wild-type and Ndst1-deficient granulocytes to Ndst1-silenced glomerular endothelial cells was found, accompanied by a decreased binding of chemokines and L-selectin. Thus, modulation of HS in the glomerular endothelial glycocalyx significantly reduced the inflammatory response in antiglomerular basement membrane nephritis.
Assuntos
Doença Antimembrana Basal Glomerular/metabolismo , Quimiotaxia de Leucócito , Células Endoteliais/metabolismo , Glicocálix/metabolismo , Heparitina Sulfato/metabolismo , Glomérulos Renais/metabolismo , Leucócitos/metabolismo , Animais , Doença Antimembrana Basal Glomerular/genética , Doença Antimembrana Basal Glomerular/imunologia , Doença Antimembrana Basal Glomerular/fisiopatologia , Doença Antimembrana Basal Glomerular/prevenção & controle , Autoanticorpos , Adesão Celular , Linhagem Celular , Quimiocinas/metabolismo , Técnicas de Cocultura , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/imunologia , Feminino , Glicocálix/imunologia , Glomérulos Renais/imunologia , Glomérulos Renais/fisiopatologia , Selectina L/metabolismo , Leucócitos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , Transdução de Sinais , Sulfotransferases/deficiência , Sulfotransferases/genética , Fatores de Tempo , TransfecçãoRESUMO
Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Transplante de Rim/efeitos adversos , Rim/metabolismo , Rim/patologia , Proteoglicanas/metabolismo , Transdução de Sinais , Aloenxertos/metabolismo , Aloenxertos/patologia , Motivos de Aminoácidos , Animais , Membrana Celular/metabolismo , Proliferação de Células , Doença Crônica , Feminino , Proteoglicanas de Heparan Sulfato/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Ligação Proteica , Ratos , Regulação para CimaRESUMO
The transient receptor potential cation channel C6 (TRPC6) is a slit diaphragm protein expressed by podocytes. TRPC6 gain-of-function mutations cause autosomal dominant focal segmental glomerulosclerosis. In acquired proteinuric renal disease, glomerular TRPC6 expression is increased. We previously demonstrated that acquired increased TRPC6 expression is ameliorated by antiproteinuric angiotensin receptor blockers and angiotensin-converting enzyme inhibitors. Vitamin D also has an antiproteinuric effect. We hypothesized that vitamin D reduces proteinuria by affecting TRPC6 expression in podocytes. Adriamycin-induced nephropathy increased TRPC6 mRNA and protein expression and induced proteinuria in rats. Treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) normalized TRPC6 expression and reduced proteinuria. In vitro, podocyte injury induced by adriamycin exposure in cultured podocytes increased TRPC6 expression. Treatment of injured podocytes with 1,25-D3 dose dependently reduced adriamycin-induced TRPC6 expression. Chromatin immunoprecipitation analysis demonstrated that the vitamin D receptor directly binds to the TRPC6 promoter. Moreover, 1,25-D3 reduced TRPC6 promoter activity in a luciferase reporter assay. In 1,25-D3-deficient 25-hydroxy-1α-hydroxylase knockout mice, TRPC6 expression was increased, accompanied by podocyte foot process effacement and proteinuria. 1,25-D3 supplementation normalized TRPC6 expression, podocyte morphology, and proteinuria in these mice. These results demonstrate that vitamin D down-regulates the enhanced TRPC6 expression in in vivo and in vitro podocyte injury, possibly through a direct effect on TRPC6 promoter activity. This TRPC6 down-regulation could contribute to the antiproteinuric effect of vitamin D.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Nefropatias/patologia , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/patologia , Canais de Cátion TRPC/genética , Vitamina D/farmacologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Animais , Imunoprecipitação da Cromatina , Regulação para Baixo/genética , Glomerulosclerose Segmentar e Focal/complicações , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Nefropatias/complicações , Nefropatias/genética , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Camundongos , Podócitos/efeitos dos fármacos , Podócitos/ultraestrutura , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Proteinúria/complicações , Proteinúria/genética , Ratos , Ratos Wistar , Receptores de Calcitriol/metabolismo , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6 , Vitamina D/análogos & derivadosRESUMO
Proteinuria is a hallmark of many glomerular diseases and an independent risk factor for the progression of renal failure. Proteinuria results from damage to the glomerular filtration barrier (GFB), which plays a critical role in size- and charge-selective filtration. The GFB consists of three layers, which is the fenestrated endothelium that is covered by the glycocalyx, the podocytes and the intervening glomerular basement membrane. Defects in one of the three layers in the GFB can lead to the development of proteinuria. Heparan sulphate (HS) is a negatively charged polysaccharide that is abundantly expressed in all layers of the GFB. HS expression in the GFB is reduced in the majority of patients with proteinuria, which is associated with an increased glomerular expression of the HS-degrading enzyme heparanase. The primary role of HS in the development of proteinuria has been challenged after the establishment of several genetically engineered mouse models with an altered HS expression that did not display development of overt proteinuria. However, in a recent study, we showed that heparanase is essential for the development of proteinuria in diabetic nephropathy, which suggests that loss of HS contributes to the development of proteinuria. Recent studies also further highlight the importance of the glomerular endothelial glycocalyx in charge-selective filtration and the development of proteinuria. This review aims to summarize our current knowledge on the role of in particular HS and heparanase in the development of proteinuria.
Assuntos
Glucuronidase/fisiologia , Glicocálix/fisiologia , Animais , Nefropatias Diabéticas/metabolismo , Feminino , Membrana Basal Glomerular/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Glomérulos Renais/metabolismo , Masculino , Podócitos/metabolismo , Proteinúria/etiologia , Proteinúria/fisiopatologiaRESUMO
BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a leading cause of steroid-resistant nephrotic syndrome. Hereditary FSGS is frequently caused by mutations in important structural podocyte proteins, including the slit diaphragm-associated transient receptor potential channel C6 (TRPC6). METHODS: In five patients with biopsy-proven autosomal-dominant FSGS from five different Dutch families, all 13 exons of TRPC6 were sequenced. Upon identification of a novel TRPC6 sequence variant, the resultant amino acid change was introduced in the wild-type TRPC6 protein and functionally tested using patch-clamp analyses and cell-surface biotinylation experiments. RESULTS: None of the previously described TRPC6 mutations were found in our cohort. In one family, we identified a novel c.524G>A sequence variant resulting in a p.Arg175Gln (R175Q) substitution in the TRPC6 protein. This sequence variant was absent in 449 control subjects and from public SNP databases. The mutation was located in the third ankyrin repeat domain (ANK3) in the cytoplasmic N-tail of TRPC6, important for protein-protein interaction and regulation of ion channel activity. Patch-clamp analyses of the mutant channel indeed showed an increased TRPC6 channel-mediated current. However, cell-surface expression of the mutant channel was not increased. CONCLUSIONS: We identified a novel TRPC6 p.Arg175Gln gain-of-function mutation that shows increased TRPC6-mediated current, which is not due to altered cell-surface expression. This is the first mutation identified in ANK3 of the TRPC6 N-tail and is most likely responsible for the late-onset autosomal dominant FSGS in this family.
Assuntos
Consanguinidade , Glomerulosclerose Segmentar e Focal/etiologia , Mutação/genética , Canais de Cátion TRPC/genética , Adulto , Idade de Início , Idoso , Sequência de Aminoácidos , Pré-Escolar , Eletrofisiologia , Família , Feminino , Seguimentos , Taxa de Filtração Glomerular , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Países Baixos , Linhagem , Prognóstico , Homologia de Sequência de Aminoácidos , Canal de Cátion TRPC6 , Fatores de TempoRESUMO
Whether renal outcomes differ between the segmental and global subclasses of diffuse proliferative (class IV) lupus nephritis is unknown. In this meta-analysis, we searched the literature in MEDLINE, EMBASE, five registries of clinical trials, and selected cohort studies and randomized, controlled trials that used the 2003 International Society of Nephrology and Renal Pathology Society classification of lupus nephritis in adult patients. Our endpoint was the composite of doubling of serum creatinine concentration or ESRD. In the eight studies included in the final analysis, the incidence of this endpoint varied between 0% and 67%. A funnel plot and Egger's test did not suggest significant heterogeneity. The meta-analysis did not support a significant difference in renal outcome between the segmental (IV-S) and global (IV-G) subclasses (relative risk for class IV-G versus IV-S, 1.08; 95% confidence interval, 0.68-1.70). Meta-regression did not suggest that ethnicity or duration of follow-up influenced the association between histologic class and renal risk. In conclusion, the rate of doubling of serum creatinine concentration or of ESRD did not differ between patients with class IV-S and those with IV-G lupus nephritis.
Assuntos
Falência Renal Crônica/etiologia , Nefrite Lúpica/complicações , Estudos de Coortes , Creatinina/sangue , Humanos , Nefrite Lúpica/sangue , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
OBJECTIVES: The objectives of this study are to analyse the long-term follow-up of a randomised controlled trial of induction treatment with azathioprine/methylprednisolone (AZA/MP) versus high-dose intravenous cyclophosphamide (ivCY) in patients with proliferative lupus nephritis (LN) and to evaluate the predictive value of clinical, laboratory and renal biopsy parameters regarding renal outcome. METHODS: 87 patients with biopsy-proven proliferative LN were treated with either AZA/MP (n=37) or ivCY (n=50), both with oral prednisone. After 2 years, renal biopsy was repeated, and all patients continued with AZA/oral prednisone. The primary study end point was sustained doubling of serum creatinine. Secondary end points included renal relapse, end-stage renal disease and mortality. RESULTS: After a median follow-up of 9.6 years, no significant differences between AZA/MP versus ivCY groups were found in the proportion of patients with sustained doubling of serum creatinine (n=6 (16%) vs n=4 (8%); p=0.313), end-stage renal disease (n=2 (5%) vs n=2 (4%); p=1.000) or mortality (n=6 (16%) vs n=5 (10%); p=0.388). Renal relapses occurred more often in the AZA/MP group (n=14 (38%) vs n=5 (10%); p=0.002, HR: 4.5). Serum creatinine, proteinuria and immunosuppressive treatment regimens at the last follow-up were comparable. Clinical and laboratory parameters at baseline and after 2 years, and renal biopsy parameters (only) at baseline predicted renal outcome. CONCLUSION: Induction treatment with ivCY was superior to AZA/MP in preventing renal relapses, but other parameters for renal function did not differ. AZA/MP can therefore serve as an alternative in patients with proliferative LN who wish to avoid gonadal toxicity of CY. Several prognostic factors of long-term renal outcome were identified.
Assuntos
Azatioprina/administração & dosagem , Ciclofosfamida/administração & dosagem , Imunossupressores/administração & dosagem , Nefrite Lúpica/tratamento farmacológico , Metilprednisolona/administração & dosagem , Adolescente , Adulto , Anti-Inflamatórios/administração & dosagem , Creatinina/sangue , Feminino , Seguimentos , Humanos , Falência Renal Crônica/mortalidade , Falência Renal Crônica/prevenção & controle , Nefrite Lúpica/sangue , Nefrite Lúpica/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteinúria/sangue , Proteinúria/tratamento farmacológico , Proteinúria/mortalidade , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
OBJECTIVES: To develop recommendations for the management of adult and paediatric lupus nephritis (LN). METHODS: The available evidence was systematically reviewed using the PubMed database. A modified Delphi method was used to compile questions, elicit expert opinions and reach consensus. RESULTS: Immunosuppressive treatment should be guided by renal biopsy, and aiming for complete renal response (proteinuria <0.5 g/24 h with normal or near-normal renal function). Hydroxychloroquine is recommended for all patients with LN. Because of a more favourable efficacy/toxicity ratio, as initial treatment for patients with class III-IV(A) or (A/C) (±V) LN according to the International Society of Nephrology/Renal Pathology Society 2003 classification, mycophenolic acid (MPA) or low-dose intravenous cyclophosphamide (CY) in combination with glucocorticoids is recommended. In patients with adverse clinical or histological features, CY can be prescribed at higher doses, while azathioprine is an alternative for milder cases. For pure class V LN with nephrotic-range proteinuria, MPA in combination with oral glucocorticoids is recommended as initial treatment. In patients improving after initial treatment, subsequent immunosuppression with MPA or azathioprine is recommended for at least 3 years; in such cases, initial treatment with MPA should be followed by MPA. For MPA or CY failures, switching to the other agent, or to rituximab, is the suggested course of action. In anticipation of pregnancy, patients should be switched to appropriate medications without reducing the intensity of treatment. There is no evidence to suggest that management of LN should differ in children versus adults. CONCLUSIONS: Recommendations for the management of LN were developed using an evidence-based approach followed by expert consensus.
Assuntos
Azatioprina/uso terapêutico , Ciclofosfamida/uso terapêutico , Gerenciamento Clínico , Glucocorticoides/uso terapêutico , Hidroxicloroquina/uso terapêutico , Imunossupressores/uso terapêutico , Nefrite Lúpica/tratamento farmacológico , Ácido Micofenólico/uso terapêutico , Adulto , Biópsia , Criança , Relação Dose-Resposta a Droga , Substituição de Medicamentos , Quimioterapia Combinada , Medicina Baseada em Evidências , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Nefrite Lúpica/diagnóstico , Nefrite Lúpica/urina , Masculino , GravidezRESUMO
The transient receptor potential channel C6 (TRPC6) is a slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Gain-of-function mutations in TRPC6 cause hereditary focal segmental glomerulosclerosis (FSGS), and several human acquired proteinuric diseases show increased glomerular TRPC6 expression. Angiotensin II (AngII) is a key contributor to glomerular disease and may regulate TRPC6 expression in nonrenal cells. We demonstrate that AngII regulates TRPC6 mRNA and protein levels in cultured podocytes and that AngII infusion enhances glomerular TRPC6 expression in vivo. In animal models for human FSGS (doxorubicin nephropathy) and increased renin-angiotensin system activity (Ren2 transgenic rats), glomerular TRPC6 expression was increased in an AngII-dependent manner. TRPC6 expression correlated with glomerular damage markers and glomerulosclerosis. We show that the regulation of TRPC6 expression by AngII and doxorubicin requires TRPC6-mediated Ca(2+) influx and the activation of the Ca(2+)-dependent protein phosphatase calcineurin and its substrate nuclear factor of activated T cells (NFAT). Accordingly, calcineurin inhibition by cyclosporine decreased TRPC6 expression and reduced proteinuria in doxorubicin nephropathy, whereas podocyte-specific inducible expression of a constitutively active NFAT mutant increased TRPC6 expression and induced severe proteinuria. Our findings demonstrate that the deleterious effects of AngII on podocytes and its pathogenic role in glomerular disease involve enhanced TRPC6 expression via a calcineurin/NFAT positive feedback signaling pathway.
Assuntos
Angiotensina II/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Podócitos/patologia , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPC/metabolismo , Antagonistas de Receptores de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Calcineurina/metabolismo , Cálcio/metabolismo , Doxorrubicina , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Nefropatias/induzido quimicamente , Nefropatias/complicações , Nefropatias/metabolismo , Nefropatias/patologia , Camundongos , Modelos Biológicos , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Proteinúria/complicações , Proteinúria/metabolismo , Proteinúria/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Renina/farmacologia , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6RESUMO
BACKGROUND: A reduced heparan sulphate (HS) expression in the glomerular basement membrane of patients with overt diabetic nephropathy is associated with an increased glomerular heparanase expression. We investigated the possible association of urinary heparanase activity with the development of proteinuria in patients with Type 1 diabetes (T1D), Type 2 diabetes (T2D), or membranous glomerulopathy (MGP) as non-diabetic disease controls. METHODS: Heparanase activity, albumin, HS and creatinine were measured in the urine of patients with T1D (n=58) or T2D (n=31), in patients with MGP (n=52) and in healthy controls (n=10). Heparanase messenger RNA (mRNA) expression in leukocytes was determined in a subgroup of patients with T1D (n=19). RESULTS: Urinary heparanase activity was increased in patients with T1D and T2D, which was more prominent in patients with macroalbuminuria, whereas no activity could be detected in healthy controls. Albuminuria levels were associated with increased urinary heparanase activity in diabetic patients (r=0.20; P<0.05) but not in patients with MGP (r=0.11; P=0.43). A lower urinary heparanase activity was observed in diabetic patients treated with inhibitors of the renin-angiotensin-aldosterone system (RAAS), when compared to diabetic patients treated with other anti-hypertensives. Additionally, urinary heparanase activity was associated with age in T1D and MGP. In MGP, heparanase activity and ß2-microglobulin excretion correlated. In patients with T1D, no differences in heparanase mRNA expression in leukocytes could be observed. CONCLUSIONS: Urinary heparanase activity is increased in diabetic patients with proteinuria. However, whether increased heparanase activity is a cause or consequence of proteinuria requires additional research.
Assuntos
Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/urina , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/urina , Membrana Basal Glomerular/patologia , Glucuronidase/urina , Heparitina Sulfato/metabolismo , Adulto , Idoso , Albuminúria/diagnóstico , Western Blotting , Estudos de Casos e Controles , Complicações do Diabetes/enzimologia , Complicações do Diabetes/etiologia , Complicações do Diabetes/urina , Feminino , Seguimentos , Glucuronidase/genética , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Sistema Renina-Angiotensina , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: alpha-Dystroglycan (alpha-DG) is a negatively charged glycoprotein that covers the surface of podocytes. A decreased glomerular expression of alpha-DG has been described in minimal change nephropathy (MCN), but not in focal segmental glomerulosclerosis (FSGS). This was suggested as a tool to distinguish these diseases. Sialic acid is a negatively charged carbohydrate extensively present on both alpha-DG and podocalyxin, which is also expressed on podocytes. Intrarenal perfusion with bacterial sialidase leads to foot process effacement and proteinuria. This is the first study on the expression of endogenous glomerular sialidase; furthermore, the expression of dystroglycan was re-evaluated. METHODS: The expression of alpha-DG and sialidase was investigated by immunofluorescence in kidney biopsies of patients with MCN (n = 5), FSGS (n = 15), proliferative lupus nephritis (LN, n = 9), membranous glomerulopathy (MG, n = 10) and normal human kidneys (NHK, n = 4). The urinary sialic acid concentration was measured using a newly developed LC-tandem mass spectrometry method. RESULTS: A 3-fold increased glomerular expression of sialidase was found in MG, accompanied with an increased urinary sialic acid concentration in two MG patients. However, we did not observe major changes in the expression of alpha-DG in patients with the above-mentioned glomerular diseases compared to NHK, also not between MCN and FSGS. CONCLUSIONS: Endogenous glomerular sialidase expression is increased in MG, which might represent a novel mechanism for the loss of negative charge in the glomerular capillary filter. The expression of dystroglycan cannot be used as a diagnostic tool to differentiate between glomerular diseases.