A nefropatia diabética (ND) é uma das principais causas de doença renal terminal. Além da lesão glomerular, o compartimento tubulointersticial é afetado no desenvolvimento da ND, não somente pela proteinúria como pelos efeitos pró-inflamatórios, profibróticos, pró-oxidantes e angiogênicos da hiperglicemia e dos produtos finais de glicação avançada (AGEs). Sabese que as concentrações de marcadores do estresse oxidativo estão aumentadas em pacientes com diabetes mellitus (DM). O sistema tiorredoxina (TXN) é um dos principais sistemas antioxidantes endógenos. A TXN é capaz de interagir com um grande número de fatores de transcrição e proteínas, tal como a Thioredoxin interacting protein (TXNIP) que tem sido reconhecida na patogênese do DM e de suas complicações. Além da TXNIP, a deficiência de tiamina, ou vitamina B1, já foi relatada em modelos experimentais de DM, concomitantemente a um aumento de seu clearance renal. Os transportadores de tiamina 1 (THTR1) e 2 (THTR2) (codificados pelos genes SLC19A2 e SLC19A3, respectivamente) são os responsáveis pela reabsorção de tiamina no túbulo proximal após sua filtração pelo glomérulo. Estudos já demonstraram que a excreção aumentada de tiamina pode ser um fator de risco para o declínio precoce da função renal em pacientes DM. Uma outra proteína de interesse é a beta 2 microglobulina (B2M), expressa em situações que cursam com inflamação, um fenômeno bem caracterizado na tubulopatia diabética. O estudo da ativação intrarenal de vias potencialmente associadas à evolução da ND em humanos é dificultada pela necessidade de biópsia renal. Recentemente o sedimento urinário tem sido utilizado na avaliação das doenças renais, tanto na tentativa de identificar biomarcadores que possam predizer o declínio da função renal, como para o melhor entendimento da patogênese dessa complicação. O objetivo deste trabalho foi estudar a participação dos genesalvo TXNIP, TXN, SLC19A2, SLC19A3 e B2M na patogênese da ND em portadores de DM1. Estudamos o RNA mensageiro (mRNA) do sedimento urinário de pacientes DM1 (n=55), portadores de glomeruloesclerose segmentar e focal (GESF, um modelo de nefropatia não diabética, n=12) e de indivíduos controles (n=11) para avaliação da expressão dos genes-alvo, e sua associação com as manifestações da ND. Também foi extraído mRNA de células linfomononucleares de pacientes DM1 (n=162) e indivíduos controles (n=26) para comparação com os resultados obtidos no sedimento urinário e foram dosadas as concentrações plasmáticas de tiamina em um subgrupos de pacientes DM1 e de indivíduos controles. Além disso, uma linhagem de células renais humanas foi tratada com altas concentrações de glicose e albumina glicada ou não glicada in vitro para avaliar se os AGEs estão implicados na alteração de expressão dos genes-alvos. Como resultado observamos (1) um aumento na expressão de TXNIP e TXN no sedimento urinário de pacientes DM1 com doença renal e associação da expressão de TXNIP com a magnitude do declínio da taxa de filtração glomerular; (2) um aumento na expressão de TXNIP e TXN nas células linfomononucleares dos pacientes DM1; (3) um aumento na expressão de SLC19A2 no sedimento urinário de pacientes DM1 com doença renal; (4) uma diminuição nas concentrações plasmáticas de tiamina nos pacientes DM1 em comparação aos controles; (5) um aumento na expressão de B2M no sedimento urinário de pacientes DM1 com doença renal e (6) um aumento na expressão de TXNIP e B2M nas células renais humanas tratadas concomitantemente com altas concentrações de glicose e albumina glicada. Os resultados do presente estudo sugerem fortemente a participação do sistema TXN e da B2M na etiopatogênese da ND / Diabetic nephropathy (DN) is a major cause of end stage renal disease. Glomerular and tubulointerstitial compartments are affected not only by proteinuria but also by the pro-inflammatory, profibrotic, pro-oxidants and pro-angiogenic effects exerted by hyperglycemia and advanced glycation end products (AGEs) in the development of DN. It is well known that concentrations of oxidative stress markers are increased in patients with diabetes mellitus (DM). The thioredoxin system (TXN) is one of the majors endogenous antioxidant systems; TXN molecule is able to interact with a large number of transcription factors and proteins such as Thioredoxin interacting protein (TXNIP), which has been recognized in the pathogenesis of DM and its complications. Deficiency of thiamine, or B1 vitamin, has been reported in experimental models of DM concomitantly with an increase in its renal clearance. Thiamine transporter 1 (THTR1), encoded by the SLC19A2 gene and Thiamine transporter 2 (THTR2), encoded by the SLC19A3 gene are responsible for thiamine reabsorption in the proximal tubule after glomerular filtration. Studies have shown that increased urinary excretion of thiamine may be a risk predictor for an early decline in kidney function in diabetic patients. Another protein of interest is beta-2-microglobulin (B2M), expressed in situations of inflammation, a well-characterized phenomenon in diabetic tubulopathy. The study of activation or inactivation of intrarenal pathways potentially associated with progression of DN in humans is complicated by the need for renal biopsy. Recently urinary sediment has been used in the evaluation of kidney diseases in an attempt to identify biomarkers that can predict kidney function decline and as a tool for a better understanding of the pathogenesis of this complication. The objective of this work was to study the participation of the target genes TXNIP, TXN, SLC19A2, SLC19A3 and B2M in the pathogenesis of DN in type 1 DM (T1D) patients. We studied the urinary sediment messenger RNA (mRNA) from patients with T1D (n=55); with focal and segmental glomerulosclerosis (FSGS, a non diabetic nephropathy model, n=12) and from control subjects (n=11) to assess the expression of the target genes and their association with the severity of DN. We also studied the mRNA expression of peripheral lymphomononuclear (PLMN) cells from T1D patients (n=162) and control subjects (n=26) in order to compare with the results obtained in the urinary sediment. Plasmatic concentrations of thiamine were also measured in a subgroup of T1D patients and control subjects. In addition, a lineage of human kidney cells was exposed to high glucose concentrations and to glycated and non-glycated albumin to evaluate if AGEs are implicated in the modulation of expression of the target genes. As a result we found that (1) TXNIP and TXN are upregulated in the urinary sediment of T1D patients with kidney disease and TXNIP is associated with the magnitude of glomerular filtration rate decline; (2) TXNIP and TXN are upregulated in PLMN cells from T1D patients; (3) SLC19A2 is upregulated in the urinary sediment of T1D patients with kidney disease; (4) T1D patients present decreased plasmatic thiamine concentrations compared to control subjects; (5) B2M is upregulated in the urinary sediment of T1D patients with kidney disease and (6) TXNIP and B2M are upregulated in human kidney cells exposed concomitantly to high glucose concentrations and glycated albumin. The results of the present study strongly suggest the participation of the TXN system and of B2M in the pathogenesis of DN. Descriptors: diabetes mellitus, diabetic nephropathies, urine, glycosylation end products, advanced, thiamine, thioredoxins, beta 2-microglobulin Diabetic nephropathy (DN) is a major cause of end stage renal disease. Glomerular and tubulointerstitial compartments are affected not only by proteinuria but also by the pro-inflammatory, profibrotic, pro-oxidants and pro-angiogenic effects exerted by hyperglycemia and advanced glycation end products (AGEs) in the development of DN. It is well known that concentrations of oxidative stress markers are increased in patients with diabetes mellitus (DM). The thioredoxin system (TXN) is one of the majors endogenous antioxidant systems; TXN molecule is able to interact with a large number of transcription factors and proteins such as Thioredoxin interacting protein (TXNIP), which has been recognized in the pathogenesis of DM and its complications. Deficiency of thiamine, or B1 vitamin, has been reported in experimental models of DM concomitantly with an increase in its renal clearance. Thiamine transporter 1 (THTR1), encoded by the SLC19A2 gene and Thiamine transporter 2 (THTR2), encoded by the SLC19A3 gene are responsible for thiamine reabsorption in the proximal tubule after glomerular filtration. Studies have shown that increased urinary excretion of thiamine may be a risk predictor for an early decline in kidney function in diabetic patients. Another protein of interest is beta-2-microglobulin (B2M), expressed in situations of inflammation, a well-characterized phenomenon in diabetic tubulopathy. The study of activation or inactivation of intrarenal pathways potentially associated with progression of DN in humans is complicated by the need for renal biopsy. Recently urinary sediment has been used in the evaluation of kidney diseases in an attempt to identify biomarkers that can predict kidney function decline and as a tool for a better understanding of the pathogenesis of this complication. The objective of this work was to study the participation of the target genes TXNIP, TXN, SLC19A2, SLC19A3 and B2M in the pathogenesis of DN in type 1 DM (T1D) patients. We studied the urinary sediment messenger RNA (mRNA) from patients with T1D (n=55); with focal and segmental glomerulosclerosis (FSGS, a non diabetic nephropathy model, n=12) and from control subjects (n=11) to assess the expression of the target genes and their association with the severity of DN. We also studied the mRNA expression of peripheral lymphomononuclear (PLMN) cells from T1D patients (n=162) and control subjects (n=26) in order to compare with the results obtained in the urinary sediment. Plasmatic concentrations of thiamine were also measured in a subgroup of T1D patients and control subjects. In addition, a lineage of human kidney cells was exposed to high glucose concentrations and to glycated and non-glycated albumin to evaluate if AGEs are implicated in the modulation of expression of the target genes. As a result we found that (1) TXNIP and TXN are upregulated in the urinary sediment of T1D patients with kidney disease and TXNIP is associated with the magnitude of glomerular filtration rate decline; (2) TXNIP and TXN are upregulated in PLMN cells from T1D patients; (3) SLC19A2 is upregulated in the urinary sediment of T1D patients with kidney disease; (4) T1D patients present decreased plasmatic thiamine concentrations compared to control subjects; (5) B2M is upregulated in the urinary sediment of T1D patients with kidney disease and (6) TXNIP and B2M are upregulated in human kidney cells exposed concomitantly to high glucose concentrations and glycated albumin. The results of the present study strongly suggest the participation of the TXN system and of B2M in the pathogenesis of DN
| Identifer | oai:union.ndltd.org:usp.br/oai:teses.usp.br:tde-11042016-115124 |
| Date | 20 January 2016 |
| Creators | Caillaud, Maria Beatriz Camargo Monteiro |
| Contributors | Giannella, Maria Lucia Cardillo Correa |
| Publisher | Biblioteca Digitais de Teses e Dissertações da USP |
| Source Sets | Universidade de São Paulo |
| Language | Portuguese |
| Detected Language | English |
| Type | Tese de Doutorado |
| Format | application/pdf |
| Rights | Liberar o conteúdo para acesso público. |
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