RESUMO
Diabetic nephropathy is the leading cause of end-stage renal disease. Because early diagnosis and treatment may prevent the complication, new tools for an early detection are needed. One of the key components of the glomerular filtration slit spanning between neighboring podocytes is nephrin. Its expression is altered in experimental models of diabetes and also in various human proteinuric diseases, including diabetes. We studied whether type 1 diabetic patients with or without nephropathy exhibit immunoreactive nephrin in the urine, reflecting early damage of the filtration barrier. Diabetic patients with normoalbuminuria (n = 40), with microalbuminuria (n = 41), and with macroalbuminuria (n = 39) and patients previously normoalbuminuric but now testing positive for microalbuminuria (newMicro, n = 39) were screened for nephrinuria with Western blotting using two affinity-purified anti-nephrin antibodies. Nondiabetic healthy subjects (n = 29) were also studied. Nephrinuria was present in 30% of normoalbuminuric, 17% of microalbuminuric, 28% of macroalbuminuric, and 28% of newMicro patients. Of female patients, 35% were nephrinuric compared with only 19% of male patients (P = 0.02). None of the control subjects was nephrinuric. In conclusion, glomerular filtration barrier may be affected in one-third of diabetic patients manifesting as early nephrinuria. Nephrinuria may have prognostic value and become a marker of susceptibility for kidney complications in diabetes.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/urina , Proteínas/metabolismo , Adulto , Western Blotting , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/urina , Feminino , Imunofluorescência , Humanos , Masculino , Proteínas de MembranaRESUMO
BACKGROUND: Patients with type 1 diabetes typically develop autoantibodies to antigens of the pancreatic islet cells including insulin, glutamic acid decarboxylase and the protein tyrosine phosphatase-related islet antigen 2 protein. Nephrin is a protein shared by the kidney glomeruli, pancreatic beta-cells and islet microendothelia. Since circulating antibodies to nephrin have been shown to cause proteinuria, we wanted to test whether such autoantibodies can be detected in diabetic patients. METHODS: We developed a radioimmunoprecipitation assay and analysed samples in a follow-up series of 66 patients with type 1 diabetes. RESULTS: A total of 24% of the patients tested positive for nephrin autoantibodies at diagnosis, whereas 23, 14 and 18% had these antibodies at 2, 5 and 10 years, respectively. During the follow-up at 16-19 years after diagnosis, 14 patients had signs of renal injury and 29% of them tested positive for nephrin autoantibodies in at least one sample. CONCLUSIONS: We conclude that a subset of patients with type 1 diabetes present with circulating autoantibodies to nephrin. However, the present data do not allow conclusions of a causative role for these antibodies in the pathogenesis of proteinuria in diabetes.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Adolescente , Autoanticorpos/química , Criança , Pré-Escolar , Estudos de Coortes , Diabetes Mellitus Tipo 1/imunologia , Feminino , Humanos , Imunoprecipitação , Lactente , Rim/patologia , Masculino , Proteínas de Membrana/sangue , Microscopia de Fluorescência , Fatores de TempoRESUMO
BACKGROUND: Many factors contribute to the pathogenesis of glomerular proteinuria, but no exact molecular mechanisms are known to date. The recently reported protein nephrin, encoded by the NPHS1 gene, appears to be crucial for the integrity of the glomerular filtration barrier. METHODS: Immunohistochemistry was used to detect possible changes in glomerular nephrin, and a new proteinuria-associated protein expression was developed in various diagnostic groups of human kidney biopsies. RESULTS: In normal control kidney, antibodies to intracellular and extracellular nephrin domain showed a typical podocyte pattern of reactivity, while the 18C7 antibody to a normally inaccessible proteinuria-associated epitope was negative. Instead, strong glomerular positivity by 18C7 was seen in membranous glomerulonephropathy, membranoproliferative glomerulonephritis, systemic lupus erythematosus and cryoglobulinemic nephritis, while with antibodies to either intracellular or extracellular nephrin domains, a down-regulation in nephrin expression pattern was shown. CONCLUSIONS: Unmasking or de novo expression of distinct glomerular proteins may be an important feature reflecting the pathophysiological events in these diseases with altered glomerular permeability, while only mild changes in the slit diaphragm protein nephrin appear to take place.
Assuntos
Nefropatias/fisiopatologia , Proteínas/genética , Proteinúria/fisiopatologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Biópsia , Epitopos/imunologia , Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Nefropatias/patologia , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Proteínas de Membrana , Dados de Sequência Molecular , Proteínas/imunologia , Proteinúria/patologiaRESUMO
BACKGROUND: The mouse, as opposed to the rat, is relatively resistant to the experimental nephrosis induced by puromycin aminonucleoside. The reason for this species specificity is not known. Apolipoprotein E (apoE)-deficient mice were used to determine whether hypercholesterolemia plays a role in inducing proteinuria. METHODS: Thirty-two mice were divided into normal and high cholesterol diet groups and then divided further into four subgroups: puromycin, puromycin+probucol, probucol and control. Urinary albumin of these mice was analyzed by nephelometry. The lipid peroxidation (LPO) end products malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were detected by immunohistochemistry, and the expression level of the glomerular slit diaphragm protein, nephrin, was studied by immunohistochemistry and real time RT-PCR. RESULTS: Overt proteinuria was induced by puromycin only in the apoE knockout mice ingesting the high cholesterol diet. The staining intensities of MDA and 4-HNE were stronger in the glomeruli of proteinuric mice compared to glomeruli of non-proteinuric mice. When serum cholesterol levels were reduced by probucol, proteinuria decreased and fewer LPO end products were seen immunohistochemically. Three and eight days after puromycin injection the level of nephrin mRNA in the kidneys of proteinuric mice decreased in comparison to the controls. Puromycin-treated mice kidneys demonstrated a clearly reduced reactivity to the nephrin antibodies. CONCLUSIONS: Hypercholesterolemia, possibly via LPO, is a prerequisite for puromycin-inducible glomerular damage in the mouse. Furthermore, nephrin protein and mRNA levels appear to be candidate markers of glomerular damage in the mouse.
Assuntos
Apolipoproteínas E/genética , Hipercolesterolemia/patologia , Nefrose/patologia , Animais , Colesterol na Dieta/farmacologia , Hipercolesterolemia/metabolismo , Imuno-Histoquímica , Peroxidação de Lipídeos , Proteínas de Membrana , Camundongos , Camundongos Knockout , Nefrose/induzido quimicamente , Nefrose/metabolismo , Inibidores da Síntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteinúria/metabolismo , Proteinúria/patologia , Puromicina , RNA Mensageiro/análiseRESUMO
The molecular mechanisms maintaining glomerular filtration barrier are under intensive study. This study describes a mutant Nphs1 mouse line generated by gene-trapping. Nephrin, encoded by Nphs1, is a structural protein of interpodocyte filtration slits crucial for formation of primary urine. Nephrin(trap/trap) mutants show characteristic features of proteinuric disease and die soon after birth. Morphologically, fibrotic glomeruli with distorted structures and cystic tubular lesions were observed, but no prominent changes in the branching morphogenesis of the developing collecting ducts could be found. Western blotting and immunohistochemical analyses confirmed the absence of nephrin in nephrin(trap/trap) glomeruli. The immunohistochemical staining showed also that the interaction partner of nephrin, CD2-associated protein (CD2AP), and the slit-diaphragm-associated protein, ZO-1alpha (-), appeared unchanged, whereas the major anionic apical membrane protein of podocytes, podocalyxin, somewhat punctate as compared with the wild-type (wt) and nephrin(wt/trap) stainings. Electron microscopy revealed that >90% of the podocyte foot processes were fused. The remaining interpodocyte junctions lacked slit diaphragms and, instead, showed tight adhering areas. In the heterozygote glomeruli, approximately one third of the foot processes were fused and real-time RT-PCR showed >60% decrease of nephrin-specific transcripts. These results show an effective nephrin gene elimination, resulting in a phenotype that resembles human congenital nephrotic syndrome. Although the nephrin(trap/trap) mice can be used to study the pathophysiology of the disease, the heterozygous mice may provide a useful model to study the gene dose effect of this crucial protein of the glomerular filtration barrier.