Orientadora : Profª. Drª. Andréa Emilia Marques Stinghen / Coorientador : Prof. Dr. Fellype de Carvalho Barreto / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 17/02/2016 / Inclui referências : f. 58-70 / Área de concentração / Resumo: Os produtos de glicação avançada (AGEs) são toxinas urêmicas com vários efeitos deletérios no organismo, envolvidos na disfunção endotelial e doença cardiovascular em pacientes com doença renal crônica (DRC). No presente trabalho, investigou-se in vitro em células endoteliais humanas, a capacidade de ligação dos AGEs ao Sevelamer, como possível estratégia terapêutica. Desta forma utilizou-se o receptor para AGEs (RAGE) e a interleucina-8 (IL-8) como biomarcadores de disfunção endotelial. Os AGEs foram preparados por glicação da albumina e caracterizados por absorbância e eletroforese em gel de poliacrilamida. Os níveis de endotoxinas dos AGEs sintetizados, foram mensurados pelo método de Limulus Amebocyte Lysate (LAL). As amostras apresentam-se livres de endotoxinas. Células endoteliais humanas foram incubadas em meio de cultura contendo AGEs (0,2 mg/mL) com ou sem Sevelamer (3%). O efeito dos AGEs sobre a viabilidade celular foi avaliado pelo ensaio de MTT. Realizou-se o ensaio de ressonância magnética nuclear (RMN), em sobrenadante celular contendo fosfato (Pi), a fim de validar o efeito quelante in vitro do Sevelamer sobre o Pi. Da mesma forma, os níveis de IL-8 foram analisados no sobrenadante por meio do ELISA. A expressão de RAGE foi avaliada por imunocitoquímica e Western blot. A propriedade quimiotática do sobrenadante celular após a exposição aos AGEs com ou sem Sevelamer, foi avaliada em monócitos humanos (Células U-937) usando o ensaio de migração com auxílio da câmara de Boyden. A exposição aos AGEs não afetou a viabilidade celular, mas induziu significativamente a expressão de RAGE quando comparados com as células controle (P<0,005); ainda, a expressão de RAGE diminuiu após tratamento com Sevelamer em relação ao tratamento somente com AGEs (P<0,05). O Sevelamer diminuiu significativamente os níveis de Pi, confirmando sua propriedade in vitro. Os níveis de IL-8 aumentaram significativamente após 6h de tratamento com AGEs quando comparado ao controle (17,14 ± 3,1 vs 8,2 ± 1,0 pg/mL; P<0,005); enquanto um aumento significativo foi observado nos níveis de IL-8 quando comparado as células tratadas somente com AGEs em relação com AGEs mais Sevelamer (17,14 ± 3,1 vs 27,6 ± 2,2 pg/mL; P<0,05). Quando os AGEs foram adicionados no meio de cultura, o sobrenadante de células endoteliais induziu a quimiotaxia de monócitos (402,3 ± 163,8%; P<0,05) , que foi reduzido (402,3 ± 163,8 vs 33,3 ± 7,4%; P<0,05) após o tratamento com Sevelamer. O tratamento com Sevelamer reduziu a expressão de RAGE e migração de monócitos e aumentou a expressão de IL-8, uma interleucina descrita como anti-inflamatória, que hipoteticamente pode levar a diminuição de moléculas de adesão. Nossos resultados sugerem que o Sevelamer é capaz de se ligar a outras toxinas urêmicas além do Pi, tais como os AGEs. Esse efeito pleiotrópico pode contribuir para a proteção do endotélio, entretanto experimentos adicionais são necessários para um melhor entendimento dos mecanismos relacionados à disfunção endotelial mediada por AGEs e o potencial papel protetor do Sevelamer. Palavras-chave: Doença renal crônica, disfunção endotelial, AGEs, Sevelamer, IL-8 e RAGE. / Abstract: Advanced glycation end products (AGEs) are uremic toxins with several pro-inflammatory effects, which have been implicated in the development of endothelial dysfunction and cardiovascular diseases in patients with chronic kidney disease (CKD). We sought to investigate the in vitro AGEs-binding capacity of Sevelamer, as a potential anti-inflammatory strategy, using human endothelial cells. For this purpose we chose the receptor for AGE (RAGE) and the interleukin-8 (IL-8) as biomarkers of endothelial dysfunction. The AGEs were prepared through the method of albumin glycation and characterized by absorbance and polyacrylamide gel electrophoresis. The AGEs endotoxin levels were measured through the Limulus Amebocyte Lysate (LAL) method. The samples were free of endotoxins. The human endothelial cells were incubated in a culture media containing AGEs (0.2 mg/mL) with or without Sevelamer (3%). The effect of AGEs on cell viability was assessed by MTT assay. A nuclear magnetic resonance (NMR) to measure the levels of phosphate in samples, was performed in order to verify Sevelamer's phosphate binder function in vitro. In addition, IL-8 levels were analyzed in cell supernatant, through enzyme linked immunosorbent assay. RAGE expression was evaluated through immunocytochemistry and Western blot. The chemotactic property of the endothelial cell supernatant after exposure to AGEs, with or without Sevelamer, was evaluated in human monocytes (U-937 cells) using Boyden chamber migration assay. Although the AGEs exposure did not affect the cell viability, it induced significantly RAGE expression when compared to control cells (P<0.005); moreover RAGE was significantly decreased after the Sevelamer treatment, if compared to the treatment with AGEs (P<0.05). The Sevelamer significantly decreased phosphate levels, therefore confirming its property in vitro. IL-8 levels significantly increased after 6h AGEs treatment when compared to control cells (17.14 ± 3.1 vs 8.2 ± 1.0 pg/mL; P<0.005); while a significant increase was observed in IL-8 levels in cells treated only with AGEs when compared to the treatment of AGEs with Sevelamer (17.14 ± 3.1 vs 27.6 ± 2.2 pg/mL; P<0.05). When AGEs were added to the culture media, the endothelial cell supernatant induced significantly monocytes chemotaxis (402.3 ± 163.8%; P<0.05) which was significantly reduced (402.3 ± 163.8 vs 33.3 ± 7.4%; P<0.05) after the Sevelamer treatment. The Sevelamer reduces RAGE expression and monocytes migration, while increasing IL-8 expression, an interleukin described as anti-inflammatory, which hypothetically could lead to the decrease in adhesion molecules. Our results suggest that the Sevelamer is capable of binding to other uremic toxins besides phosphate, such as the AGEs. This pleiotropic effect may contribute to its role on the endothelium protection. Additional experiments are necessary for a better understanding of the mechanisms related to the AGEs mediated endothelial dysfunction and the Sevelamer potential protective role. Keywords: Chronic kidney disease, endothelial disfuncion, AGEs, Sevelamer, IL-8 e RAGE.

Bakgrund: Det finns läkemedel som ger ökad risk för vitamin D-brist. En kartläggningsstudie av hur patientgrupper med olika riskläkemedel hanteras i Landstinget i Kalmar län har inte gjorts tidigare. En sådan studie är önskvärd, för att få en nulägesanalys och öka medvetenheten bland hälso-och sjukvårdspersonal om hur läkemedel påverkar D-vitaminstatus. Syfte: Syftet var att undersöka omfattningen av ordinerade riskläkemedel hos samtliga patienter, samt hos patientgruppen ≥75år och hur dessa handläggs avseende ordination av supplementering (samtidig behandling med läkemedel innehållande vitamin D3 / kalcium och vitamin D3,), provtagning och analysresultat avseende kalcidiol i serum. Metod: Riskläkemedel för vitamin D-brist identifierades. Inklusionskriterier för studien var patienter med de fördefinierade riskläkemedlen med ordinationer under 2012 och/eller 2013. Populationen som ingick i studien var patienter i Landstinget i Kalmar län. Data hämtades från patientdatasystemet Cambio Cosmic. Utifrån dessa data analyserades förekomst av supplementering, provtagning för S-25(OH)D kalcidiol och analyssvar. Materialet strukturerades efter respektive läkemedel avseende kön och ålder (≥75 år.) Resultat: 9118 individer ordinerades något av riskläkemedelen orlistat, sevelamer, kolestyramin, kolestipol, efavirenz, prednison, prednisolon, fenytoin, fenobarbital eller karbamazepin. 31% av patienterna som ordinerats riskläkemedel var ≥75 år. Totalt behandlades 22% med supplementationspreparat av de som ordinerats riskläkemedel. I åldersgruppen ≥75 år behandlades 43% med supplement. 61% av de supplementerade patienterna var ≥75 år. Totalt provtogs enbart 4,1% avseende kalcidiol av de som ordinerats riskläkemedel. Av dessa hade 37% av de provtagna en bristnivå av S-25(OH)D (≤50 nmol/L) vid första provtillfället. 57% av de som visade brist supplementerades. Endast 8,3% av de som hade en brist följdes upp med ytterligare en eller flera provtagningar. Av patienterna ≥75 år provtogs 4,1% av patienterna i åldersgruppen. Detta innebär att 31% av de som provtogs var ≥75år. 39% led brist. Av de patienter ≥75 år som hade led brist supplementerades 65% av individerna. Konklusion: Det finns förbättringsutrymme för implementering av kunskap kring riskläkemedel i Landstinget i Kalmar län. / Introduction: Some drugs increase the risk for vitamin D deficiency. To date, no survey has been performed in Kalmar County about how patients medicated with risk pharmaceuticals are handled. Such a survey would be desirable in order to increase knowledge among healthcare workers about how substances interfere with vitamin D status. Aim: The aim was to examine the extent to which risk pharmaceuticals are prescribed among all patients and among patients aged ≥75, and to investigate how these patients are treated with respect to supplementation (drugs containing vitamin D3 / Calcuim and vitamin D3), blood sampling and analysis of serum calcidiol levels. Method: Drugs interfering with vitamin D were identified. Inclusion criteria for the survey were patients that had been prescribed the pre-defined risk pharmaceuticals during 2012 and/ or 2013. The surveyed population was residents in Kalmar County. Data were collected from the patient database Cambio Cosmic. From these data analyzes were made about the occurrence of: supplementation, sampling of S-25(OH)D calcidiol and test results. The collected data were arranged by substance, gender and age (elderly aged ≥75). Results: 9118 patients were prescribed at least one of the risk pharmaceuticals; orlistat, sevelamer, cholestyramine, colestipol, efavirenz, prednisol, prednison, phenytoin, phenobarbital or carbamazepine. 31% of these were aged ≥75. Overall, 22% of the patients were prescribed supplements. Out of these, 61% were elderly. Among the patients that had been prescribed risk pharmaceuticals a minority, 4,1% of the patients were sampled for calcidiol. 37% of these had deficiency in S-25(OH)D (≤50 nmol/L). 8.3% of patients with a vitamin D deficiency were sampled more than once. Out of the patients with deficiency 57% were treated with supplements. Out of the elderly patients prescribed risk pharmaceuticals, 4.1% of the patients were tested for calcidiol. This means that 31% of the tested patients were aged ≥75 and 39% of these had a deficiency. Out of the elderly patients with deficiency, 65% were treated with supplements. Conclusion: In Kalmar County, much can be done in order to better implement the existing knowledge about drugs that increase the risk for vitamin D deficiency. / Betydelsen av bra D-vitaminstatus för äldres hälsa – Hur stor är förekomsten av D-vitaminbrist hos äldre i Kalmar län och hur påverkar läkemedel D-vitaminstatus