Diabetic nephropathy is a common diabetic microvascular disease with a prevalence of about 10% to 42%. Research has shown that neutrophil gelatinase-associated lipocalin (NGAL) levels would increase rapidly in the urine and blood of patients with acute kidney failure. NGAL may represent an early and predictive kidney injury biomarker due to the increase of NGAL occurs earlier than that of molecules (creatinine, cystatin C and £]2-microglobulin) for traditional assessment of renal injury in renal disease samples. To evaluate the association of plasma level of NGAL and £]2-microglobulin with diabetic nephropathy, this study was performed on 21 diabetic patients without nephropathy as the control group and 21 patients with diabetic nephropathy stage 2, 26 patients with stage 3, 9 patients with stage 4 and 16 patients stage 5 as the study group. Collection of blood and measurements of all cases were approved by the ethical committee. The results indicate that the levels of blood urea nitrogen (BUN), creatinine, NGAL, and £]2-microglobulin of study group were significantly higher than control group (P<0.001), while the glomerular filtration rate (GFR) was significantly lower than the control group (P<0.001). Linear regression analysis show that NGAL was positively correlated with white blood cells, BUN, creatinine, £]2-micrglobulin and negatively correlated with GFR; and £]2-micrglobulin was positively correlated with BUN, creatinine, NGAL and negatively correlated with GFR. All results indicate that plasma NGAL levels in diabetic nephropathy were positively correlated with renal function parameters, and closely correlated with kidney injury, suggesting that NGAL may play an important role in the progression of diabetic nephropathy.

Submitted by Marcia Clelia Leite Marcellino (marcia.clelia@terra.com.br) on 2018-06-11T02:33:02Z No. of bitstreams: 1 TESE IMPRESSÃO.pdf: 1297621 bytes, checksum: 72d61d4f45d3f68457e648dba38bd91f (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-06-13T16:42:35Z (GMT) No. of bitstreams: 1 marcellino_mcl_dr_bot.pdf: 1297621 bytes, checksum: 72d61d4f45d3f68457e648dba38bd91f (MD5) / Made available in DSpace on 2018-06-13T16:42:35Z (GMT). No. of bitstreams: 1 marcellino_mcl_dr_bot.pdf: 1297621 bytes, checksum: 72d61d4f45d3f68457e648dba38bd91f (MD5) Previous issue date: 2018-03-29 / A nefropatia diabética é fator de risco para aumento da morbimortalidade em pessoas submetidas à cirurgia. A investigação por tratamentos capazes de exercer proteção renal durante procedimentos anestésico- cirúrgicos tornam-se relevantes. Este estudo avaliou a glicemia, a variação ponderal, a concentração sanguínea de ureia e creatinina, a proteinúria, a microscopia eletrônica dos rins com e sem isquemia e reperfusão, e a proteção renal de ratos Wistar com nefropatia diabética experimental, tratados previamente com a infusão dos frutos da Momordica charantia L.. Utilizamos 26 ratos Wistar, divididos em: Grupo Controle (n=10); Grupo Diabético sem tratamento (n=8) e Grupo Diabético tratados com Momordica charanthia L. (n=8). A determinação da glicemia de ambos os grupos foi feita com glicosimetro e o peso foi mensurado em balança digital no 1º, 15º e 30º dia do experimento. A dosagem da proteinúria foi feita em urina de 24 horas. Previamente a eutanásia, foi realizada a manobra da isquemia por 30 minutos, seguida da reperfusão por 15 minutos, no rim esquerdo, simulando a lesão renal ocorrida em cirurgias. Os rins isquêmicos e não isquêmicos foram encaminhados para microscopia eletrônica. A obtenção do sangue para dosagem de ureia e creatinina foi feita por punção cardíaca. Os resultados obtidos evidenciaram redução significativa da glicemia (p<0,001- Teste Friedman p<0,05) no 15ª dia, nos animais diabéticos tratados com a planta, Não foi evidenciada alterações significativas no peso dos animais. A Momordica charantia L. reduziu significativamente a concentração de creatinina (p=0,022 – Teste T-Student p<0,05) e a proteinúria (p<0,001 – Teste T-Student p<0,05), mas não interferiu na uremia dos animais diabéticos. Na microscopia eletrônica evidenciou-se nos rins isquêmicos e não isquêmicos do grupo diabético tratado, a preservação da membrana basal, podócitos e pedicelos. Estas estruturas foram lesionadas no grupo diabético sem tratamento. Conclui-se que a Momordica charantia L. mostrou-se eficaz na redução da glicemia, no 15º dia de experimentação; reduziu a creatinina e a proteinúria em relação ao grupo diabético sem tratamento e preservou a integridade de estruturas glomerulares, sugerindo renoproteção frente à hiperglicemia persistente e a manobra de isquemia e reperfusão renal. / Diabetic nephropathy is a risk factor for increased morbidity and mortality in people undergoing surgery. The investigation of treatments capable of exerting renal protection during anesthetic-surgical procedures becomes relevant. Diabetic nephropathy is a risk factor for increased morbidity and mortality in people undergoing surgery. The investigation of treatments capable of exerting renal protection during anesthetic-surgical procedures becomes relevant. This study evaluated glycemia, weight variation, blood urea and creatinine concentration, proteinuria, electron microscopy of the kidneys with and without ischemia and reperfusion, and renal protection of Wistar rats with experimental diabetic nephropathy, previously treated with the infusion of the fruits of Momordica charantia L. We used 26 Wistar rats, divided into: Control Group (n = 10); Untreated Diabetic Group (n = 8) and Diabetic Group treated with Momordica charanthia L. (n = 8). The determination of glycemia was done with glycosimetre and the weight was measured in digital scale on the 1st, 15th and 30th day of experimentation. The proteinuria was measured in 24-hour urine. Before euthanasia, the maneuver of the ischemia was performed for 30 minutes, followed by reperfusion for 15 minutes in the left kidney, simulating the renal injury that occurred in surgeries. The ischemic and non-ischemic kidneys were referred for electron microscopy. The blood obtained for urea and creatinine dosing was done by cardiac puncture. The results obtained evidenced a significant reduction of glycemia (p <0.001 Friedman test p <0.05) on the 15th day, in the diabetic animals treated with the plant. No significant changes were observed in the weight of the animals. Momordica charantia L. significantly reduced creatinine concentration (p = 0.022 - Student's T-test p <0.05) and proteinuria (p <0.001 - Student's T-test p <0.05) but did not interfere with uremia of diabetic animals. Electron microscopy revealed preservation of the basement membrane, podocytes and pedicels in the ischemic and non-ischemic kidneys of the treated diabetic group. These structures were injured in the diabetic group without treatment. It was concluded that Momordica charantia L. was effective in the reduction of glycemia, on the 15th day of experimentation; reduced creatinine and proteinuria in relation to the untreated diabetic group and preserved the integrity of glomerular structures, suggesting renoprotection against persistent hyperglycemia and renal ischemia and reperfusion maneuver.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21833号 / 農博第2346号 / 新制||農||1068(附属図書館) / 学位論文||H31||N5205(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 菅原 達也, 教授 澤山 茂樹, 教授 佐藤 健司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20263号 / 医博第4222号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 小川 修, 教授 福原 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Résumé : La néphropathie diabétique (ND) est la principale cause d’insuffisance rénale de stade terminal en Amérique du Nord. Les podocytes, cellules épithéliales hautement spécialisées du glomérule, supportent et maintiennent les mécanismes de filtration glomérulaire. Des biopsies de reins de patients diabétiques ont montré que le nombre de podocytes est significativement réduit chez les patients avec un diabète récent. Néphrine est une protéine transmembranaire qui a été démontrée comme ayant un rôle majeur dans le maintien de l’intégrité de ces cellules. Une diminution de l’expression de néphrine est observée chez les personnes atteintes de la ND. Des études ont démontré que la phosphorylation en tyrosine de néphrine était impliquée dans la régulation de l’inhibition des voies de l’apoptose et le remodelage du cytosquelette d’actine. Notre laboratoire a publié que l’expression de la tyrosine phosphatase SHP-1 était augmentée dans les podocytes exposés à des concentrations élevées de glucose (HG). Les résidus tyrosines de néphrine sont contenus dans des séquences pouvant être reconnues par SHP-1. Notre hypothèse est que SHP-1 interagit avec néphrine, et que l’augmentation de l’expression de SHP-1 en condition d’hyperglycémie et de diabète viendrait déréguler les voies de signalisation de néphrine, contribuant aux dommages des podocytes dans la maladie. Des coimmunoprécipitations dans des podocytes montrent une interaction entre SHP-1 et néphrine, qui est augmentée en condition HG. Cette augmentation en HG était associée à une baisse des niveaux de phosphorylation de néphrine. La surexpression de la forme inactive de SHP-1 dans les podocyte rétablie les niveaux de phosphorylation de néphrine en condition HG. Dans un modèle de surexpression avec des cellules HEK, la surexpression de SHP-1 diminue les niveaux de phosphorylation des tyrosines 1176/1193 et 1217, qui sont associées au remodelage de l’actine. Des coimmunoprécipitations avec des mutants de néphrine montrent que les tyrosines 1114 et 1138 sont essentielles pour l’interaction de SHP-1 avec néphrine. Dans un modèle murin de diabète de type 1, une diminution de l’expression et de la phosphorylation de néphrine sont observée comparativement aux souris de type sauvage. Ces diminutions sont associées avec une augmentation de l’expression de SHP-1. En conclusion, l’augmentation de l’expression de SHP-1 en condition d’hyperglycémie réduit les niveaux de phosphorylation en tyrosine de néphrine et vient potentiellement inhiber ses voies de signalisation dans le diabète, contribuant à la dysfonction podocytaire et à la néphropathie diabétique. / Abstract : Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in North America. Podocytes are highly specialized epithelial cells involved in the glomerular filtration process. Morphometric observation from kidney biopsies of diabetic patients showed a significant reduction in the number of podocytes in patients with short duration of diabetes before the apparition of microalbuminuria. Nephrin, a transmembrane protein found in the slit diaphragm, has been found to play a key role in the integrity of the podocytes. Clinical observations indicated that nephrin expression was reduced in kidney biopsy of diabetes patients. Recent studies have shown that phosphorylation of tyrosine residues of nephrin participate in intracellular pathways regulating actin dynamics and podocyte survival. Our laboratory has recently published that the expression of the tyrosine phosphatase SHP-1 is elevated in podocytes exposed to high glucose concentrations (HG). Nephrin contains sequences that are known to be potential target for SHP-1. Our hypothesis is that SHP-1 can interact with nephrin, and the increase of SHP-1 expression in diabetic nephropathy deregulates nephrin-mediated pathways, contributing to podocyte’s damage in the disease. Coimmunoprecipitation experiments show an interaction between SHP-1 and nephrin which is increased in podocytes exposed to HG. Overexpression of the inactive form of SHP-1 in podocytes exposed to HG restores nephrin phosphorylation. In HEK cells, overexpression of SHP-1 reduces nephrin phosphorylation specifically on tyrosine 1176/1193 and 1217, which regulates actin dynamics. Coimmunoprecipitation experiments with nephrin mutants show that tyrosine 1114 and 1138 are essentials to the interaction between SHP-1 and nephrin. In a type 1 diabetic murine model, a reduction of the expression and phosphorylation levels of nephrin are observed. Both reductions are associated with an increase in SHP-1 expression. In conclusion, diabetes triggered SHP-1 expression in podocytes which reduces nephrin tyrosine phosphorylation and potentially inhibits nephrin signaling in diabetes, contributing to podocytes dysfunction in diabetic nephropathy.

La néphropathie diabétique (ND) est l'une des complications graves du diabète. Elle affecte environ 30% des patients diabétiques. La microalbuminurie est actuellement l'élément diagnostique principal de la survenue de la ND mais manque de spécificité et précocité. Plusieurs études ont été consacrées à la recherche de nouveaux biomarqueurs (BM) de la ND par des approches protéomiques. Nous avons montré dans la phase initiale de notre travail que si de nombreux candidats BM avaient été identifiés, leur nature ne faisait pas consensus et que de nombreuses études, de par leur conception, ne pouvaient identifier de BM plus précoces que l'albumine. Partant de ce constat, nous avons sélectionné une cohorte originale de diabétiques de type 1 normoalbuminuriques considérés à risque de développer la ND, sur la base de l'apparition d'une microalbuminurie consécutive à un test d'effort. Une cohorte contrôle a été aussi constituée. Dans la première partie de notre travail, nous avons comparé par électrophorèse bidimensionnelle les protéomes urinaires des patients des deux coho rtes. Les BM candidats ont été ensuite identifiés par spectrométrie de masse. L'analyse fonctionnelle de ces protéines a montré que certaines sont impliquées dans la cascade de la coagulation et les mécanismes de dysfonctionnement endothélial. Le caractère diagnostique de ces protéines a été validé dans les mêmes cohortes de patients par des expériences de Western-blot. La compréhension de la nature et de la fonction physiologique des BM candidats identifiés nous a permis de mieux appréhender les mécanismes moléculaires de la pathogénèse de la ND et d'identifier des candidats biomarqueurs plus précoces que l'albumine. Ces résultats sont présentés sous la forme d'un projet d'article .La deuxième partie de notre travail expérimental est constituée d'études préliminaires visant à la recherche de protéines spécifiques urinaires, néphrine et isoformes de l'adiponectine, afin d'évaluer leur potentiel diagnostique. 1 Ben Ameur R. et al Proteomic approaches for discovering biomarkers of diabetic nephropathy. Nephrol Dial Transplant 25, 2866-752 Ben Ameur R. et al Identification of early candidate biomarkers for diabetic nephropathy by urine proteomic analysis. To be submitted / Diabetic nephropathy (DN) is one of the most serious complications of diabetes. It affects about 30% of diabetic patients. Microalbuminuria is currently the main available marker for DN risk, but has inadequate specificity and precocity. Several published studies intended to research new biomarkers (BM) of DN by proteomic approaches. We have shown1 that, if several candidate BM were claimed, there was no consensus about their nature and that a number of studies could not identify BM earlier than albumin because of the study design. Thus, we have selected an original cohort of type 1 diabetic patients considered at risk of developing DN, on the basis of urinary albumin excretion after an exercice test. A control cohort was also enrolled. Using 2D gel electrophoresis we compared the urinary proteomes of patients from both cohorts. Then, candidate BM were identified by mass spectrometry. Functional analysis of these proteins showed that some are involved in the coagulation cascade and in mechanisms of endothelial dysfunction. The diagnostic potential of these proteins was validated by Western blotti ng. The nature and physiological function of candidate biomarkers allowed to better understand the molecular pathogenic mechanisms of DN. Results from this part of the work are shown in the form of an article2. Preliminary studies to assess the diagnostic potential research of specific urinary proteins (nephrin and different isoforms of adiponectin) are also presented.1 Ben Ameur R. et al Proteomic approaches for discovering biomarkers of diabetic nephropathy. Nephrol Dial Transplant 25, 2866-752 Ben Ameur R. et al Identification of early candidate biomarkers for diabetic nephropathy by urine proteomic analysis. To be submitted

Introdução: A lesão do podócito exerce um papel crítico na nefropatia diabética (ND) e é um fator preditivo de albuminúria patológica e progressão da doença. Neste estudo foi avaliada a expressão gênica de proteínas associadas ao podócito na urina de pacientes diabéticos em diferentes estágios da ND e em indivíduos com pré diabetes. Material e Métodos: Foram estudados 67 pacientes diabéticos com normo (n=34), micro (n=14) ou macroalbuminúria (n=19), dezenove indivíduos pré diabéticos e 15 controles saudáveis. O RNAm de nefrina, podocina, podocalixina, sinaptopodina, Transient Receptor Potential Cation Channel 6 (TRPC6), alfa actinina-4 e TGF1 foi quantificado por PCR em tempo real (2-ΔΔCt) em células do sedimento urinário. A expressão dos genes alvo do podócito foi correlacionada com albuminúria, controle glicêmico e função renal. O desempenho diagnóstico dos genes para albuminúria patológica foi determinado por curva ROC, e o seu efeito independente sobre esse desfecho foi avaliado por análise de regressão de Poisson. Resultados: O RNAm na urina dos genes alvo foi significativamente maior nos pacientes diabéticos em comparação aos não diabéticos, exceto de sinaptopodina e TGFβ1. A expressão de nefrina foi mais elevada nos indivíduos diabéticos micro e macroalbuminúricos comparado aos controles (p=0,04 e p<0,001 respectivamente), pré diabéticos (p<0,05) e normoalbuminúricos (p<0,05). Embora sua expressão tenha sido maior do que nos não diabéticos, os genes TRPC6, podocalixina e alfa actinina-4 não discriminaram os estágios da ND. A correlação da expressão dos genes com albuminúria e hemoglobina glicada foi estatisticamente significativa. Pacientes pré diabéticos tiveram expressão gênica semelhante aos controles. Na análise multivariada, apenas o gene da nefrina foi preditivo de albuminúria patológica. 6 Conclusões: A expressão das proteínas associadas ao podócito na urina foi maior nos pacientes diabéticos, mas não houve correlação direta do RNAm dos genes com níveis crescentes de albuminúria, exceto de nefrina. O gene da nefrina foi o único que discriminou os diferentes estágios da ND e foi preditivo de albuminúria patológica, mas a podocalixina e o TRPC6 também se correlacionaram com albuminúria e controle glicêmico. Neste estudo preliminar não se identificou aumento da expressão gênica das proteínas do podócito na urina em indivíduos com pré diabetes. / Introduction: Podocyte damage plays a critical role in the development of diabetic nephropathy (DN). The present study evaluated gene expression of podocyte-associated proteins in urine of pre-diabetic and diabetic patients at different stages of DN. Material and Methods: We studied 19 pre-diabetic patients, 67 diabetic patients with normo (n = 34), micro (n = 15), or macroalbuminuria (n = 19), and 15 healthy controls. Levels of mRNA of nephrin, podocin, podocalyxin, synaptopodin, transient receptor potential cation channel 6 (TRPC6), alpha-actinin-4, and TGF-1 were quantitatively measured by real-time polymerase chain reaction in urinary sediment. Gene expression was correlated with albuminuria, glycemic control, and renal function. The diagnostic performance of the genes for detecting pathological albuminuria was assessed by the receiver operating characteristic (ROC) curve and Poisson regression. Results: The mRNA expression of target genes in urinary sediment was significantly higher in diabetic compared to pre-diabetic patients and controls. Levels of nephrin were higher in diabetic patients with micro or macroalbuminuria than controls (p= 0.04 and p<0.001, respectively), pre-diabetic (p<0.05), and diabetic patients with normoalbuminuria (p<0.05), and increased with increasing rates of albuminuria. Gene expression was similar in pre-diabetic patients and controls. There was a significant positive correlation of gene expression with albuminuria and glycated hemoglobin. In the multivariate analysis, only nephrinuria predicted pathological albuminuria. Conclusions: The expression of podocyte-associated proteins in urine was higher in diabetic patients, but only nephrin correlated with increasing albuminuria and predicted 8 pathological albuminuria. This preliminary study did not find increased gene transcription in pre-diabetic patients.

A hiperglicemia é um fator de risco na progressão da ND, que associada à atividade do SRA sistêmico e/ou intrarrenal e à síntese de ET1, resulta em perda da função renal. Objetivo: Investigar o papel da hiperglicemia e a relação com a síntese de Ang II e ET-1 no tecido renal no início da ND. Métodos: Ratos Wistar foram organizados em controle; diabéticos via STZ; tratados com losartan e outros com BQ123. Foram avaliados os parâmetros metabólicos e de função renal. Resultados: O losartan preveniu o efeito da STZ na expressão de renina medular, injúria tubular, expressão de desmina e de RNAm para TNF&#945; e Nox4. O BQ123 não alterou o efeito da STZ em nenhum dos parâmetros estudados. Conclusão: A hiperglicemia tem predominância na injúria renal nos primeiros estágios da ND. Nessa condição, a Ang II sistêmica e/ou intrarrenal via receptor AT1 amplia os efeitos da hiperglicemia nos eventos iniciais da ND. / Hyperglycemia and a risk factor in the progression of ND, which associated with the RAS activity of systemic and/or intrarrenal and the synthesis of ET-1, results in loss of renal function. Aim: To investigate the role of hyperglycemia and a relationship with a synthesis of Ang II and ET-1 in the renal tissue at the beginning of DN. Methods: Wistar rats were organized in control; Diabetics via STZ; Treated with losartan and others with BQ123. Metabolic and renal function parameters were evaluated. Results: Losartan prevented the effect of STZ on the expression of medullary renin, tubular injury, desmin and mRNA expression for TNF&#945; and Nox4. BQ123 did not alter the STZ effect in any of the parameters studied. Conclusion: Hyperglycemia has a predominance of renal injury in the early stages of ND. In this condition, Ang II systemic and/or intrarenal via the AT1 receptor amplifies the effects of hyperglycemia in the initial events of ND.

Background/aim. The incidence of microvascular complications has been reported to be unchanged the last decades. However, in randomized clinical trials it has been shown that improved metabolic control can reduce the development of long term complications. It has been debated whether it is possible to achieve the same results in an unselected population. In a previous study we found a decreased incidence of overt nephropathy, but unchanged incidence of severe laser treated retinopathy in a population of patients with type 1 diabetes diagnosed in childhood. The aim of the present study was to investigate the incidence 10 years later in the same population and to analyse the importance of possible risk factors. In another previous study we found a high prevalence of subclinical neuropathy among young diabetic patients despite intensive insulin therapy since diagnosis. The aim of the present study was to examine if intensive treatment is more effective in preventing early diabetic complications other than neuropathy. The incidence of type 1 diabetes has doubled in Sweden the last decades. The reason must be environmental factors. These, as well as more intensive insulin regimens from onset of diabetes, might also lead to different disease process. We wanted to analyse if clinical characteristics at onset had changed the last 25 years and if there was any secular trend of C-peptide secretion. We also intended to investigate if longer persistence of C-peptide secretion could be of importance for prevention of long term complications. Methods. The whole study population consisted of all 478 patients with type 1 diabetes diagnosed before the age of 15 during the years 1961 - 2000, living in the catchment area of the Paediatric Clinic, University Hospital, Linköping, Sweden. For the statistical analysis the population was divided into five–year cohorts according to time of onset of diabetes. The cumulative proportion of severe retinopathy and overt nephropathy in 269 patients with onset of diabetes 1961 - 1985 was computed with survival analysis. Multivariable regression models were used to analyse the importance of metabolic control, diabetes duration, blood pressure, smoking, BMI, lipids and persisting C-peptide secretion. The prevalence of all grades of retinal changes, nephropathy and neuropathy, defined as abnormal nerve conduction, was estimated in the late 1990s in a subgroup of 80 children and adolescents with mean 13 years of diabetes duration. Clinical characteristics at onset, duration of partial remission and regularly measurements of fasting and stimulated C-peptide secretion the first five years after onset were analysed in 316 patients with onset of diabetes 1976 - 2000. Results. The cumulative proportion of severe laser treated retinopathy showed a significant declining trend the last decades. The decrease was significant between the oldest cohort with diabetes onset 1961 - 1965 and the cohorts with diabetes onset 1971 - 1975 and 1976 - 1980. The cumulative proportion of overt nephropathy also declined with a significant decrease between the oldest cohorts and all the following cohorts. After 25 years of diabetes duration it was 30% and 8% in the two oldest cohorts respectively and remained largely unchanged after 30 years. Diabetes duration and long term HbA1c were the only significant independent risk factors for both retinopathy and nephropathy. The risk of overt nephropathy increased substantially when HbA1c was above 8.5%, while the risk of severe retinopathy increased already when HbA1c exceeded 7.5%. The prevalence of neuropathy was 59%, of retinopathy 27% and of nephropathy 5% in the population of young patients after mean 13 years of diabetes duration. During the last 25 years the clinical characteristics at onset were unchanged as well as duration of partial remission and magnitude and persistence of C-peptide secretion. Conclusions. In this unselected population the cumulative proportion of severe retinopathy and overt nephropathy decreased over the last decades. Diabetic nephropathy has probably been prevented and not just postponed. Good glycaemic control was the most important factor to avoid complications, with the necessity of a lower level of HbA1c to escape retinopathy than nephropathy. Intensive insulin regimens from diabetes onset was not sufficient to entirely escape early diabetic complications after mean 13 years of diabetes duration, even if the prevalence of retinopathy and especially nephropathy was lower than usually reported. The clinical picture at onset of diabetes was unchanged the last 25 years. There was no secular trend of partial diabetes remission or C-peptide secretion during the first years after diagnosis.