| Abstract | Uvod: Tlr4 predstavlja receptor za endogene alarmine koji se pojačano proizvode tijekom transplantacije. Ishemijsko reperfuzijska ozljeda povezuje se s proizvodnjom alarmina. Opisani su polimorfizmi unutar gena tlr4 koji su povezani s smanjenim odgovorom na alarmine. Cilj istraživanja: U ovom istraživanju ispitali smo povezanost polimorfizma (rs4986790(Asp299Gly) i rs4986791 (Thr399Il) tlr4 gena i izražaja tlr4 na perifernim monocitima u bolesnika s presađenim bubregom i u zdravih kontrola. Ispitali smo utjecaj ispitivanih parametra na funkciju presađenog bubrega te na pobol bolesnika s presađenim bubregom. Nacrt studije: Presječno istraživanje. Ispitanici i metode: U studiju je bilo uključeno 120 ispitanika podjeljenih u dvije skupine. Skupinu bolesnika činilo je 70 bolesnika s presađenim bubregom i 50 zdravih kontrola. Izolacija DNA učinjena je iz pune krvi na EDTA antikoagulansu primjenom komercijalnih kolona za izolaciju G-spinTM Genomic DNA Extraction Kit (Intron Biotechnology, Seul, Južna Koreja). Polimorfizmi su analizirani PCR metodom. U skupini ispitanika analizirani su podatci vezani uz funkciju presađenog bubrega (podatci darivatelja, primatelja, podudarnost, lijekovi, trajanje hladne ishemije i komplikacije. Statistička analiza učinjena je pomoću SPSS 17.0, uz statističku značajnost ako je p<0.05. Rezultati: Nije bilo razlike u distribuciji divljih i mutiranih alela između skupina ispitanika. Asp299Gly polimorfizam prisutan je u 14,3% transplantiranih bolesnika, dok se Thr399Ile nalazi u 11,3%. U svih bolesnika analizirali smo izražaj TLR-4 i CD14 na monocitima periferne krvi. Značajno je manji postotak monocita (6,4%) (Mann Whitney test, p=0,001) u kontrolnoj skupini, u odnosu na ispitanike s presađenim bubregom (7,3%). Izražaj tlr4 i CD14 na perifernim monocitima značajno je viši u transplantiranih ispitanika. Izražaj tlr4 ovisi o prisutnosti ispitivanog polimorfizma i o vrsti imunosupresijske terapije. Bolesnici koji uzimaju tacrolimus imaju statistički značajno višu razinu tlr4 MFI. U skupini transplantiranih ispitanika s AA - divljim tipom za Asp299Gly (rs4986790) značajno je viši izražaj tlr4 (Mann Whitney test, p=0,004), jednako tako i za CC divlji tip za Thr399Ile(rs4986791) gdje iznosi 107,1 (interkvartilnog raspona 96,2 – 119,9) (Mann Whitney test, p=0,008). Vrijeme proteklo od transplantacije nije imalo utjecaj na izražaj tlr4 receptora. Ispitali smo utjecaj genotipa na biokemijsko hematološke parametre u krvi u ispitanika s presađenim bubregom. Uočavaju se značajno niže vrijednosti eritrocita (Kruskal Wallis test, p=0,037), klirensa kreatinina (Kruskal Wallis test, p=0,019) i hemoglobina (Kruskal Wallis test, p=0,009) u skupini AA-divljeg tipa rs4984790, dok su najveće izmjerene vrijednosti glukoze u skupini AG-heterozigot. Vrijednosti ureje su značajno više kod AA-divljeg tipa u CC-divljem tipu rs4986791 značajno su najniže vrijednosti eritrocita (Kruskal Wallis test, p=0,027), klirensa kreatinina (Kruskal Wallis test, p=0,030) i hemoglobina (Kruskal Wallis test, p=0,049) u trenutku protočne. Srednja vrijednost trajanja hladne ishemije je 14,2 sata (interkvartilnog raspona 12,5 – 16,8 sati). Srednja vrijednost trajanja hladne ishemije u satima obrnuto je proporcionalna klirensu kreatinina u trenutku protočne (Spearmanov koeficijent korelacije ρ= -0,394, p=0,004). Nismo utvrdili postojanje povezanosti između polimorfizama i akutnih komplikacija presađivanja bubrega kao što je odgođena funkcija presatka ili pak s komorbiditetima nakon transplantacije. Zaključak: Nema razlike u distribuciji genotipa prema skupinama ispitanika. Transplantirani bolesnici imaju veći izražaj tlr4 na perifernim monocitima. Genotip utječe na izražaj tlr4 tako što mutirani aleli uzrokuju slabiju ekspresiju tlr4 na monocitima. Osim toga nosioci mutiranih alela imaju bolju funkciju bubrega mjerenu klirensom kreatinina u trenutku protočne citometrije ukazjući na moguću zaštitnu ulogu mutiranih alela. |
| Abstract (english) | Introduction: Tlr4 is the receptor for endogenous alarmine which is overly expressed during kidney transplantation. Ischemia and reperfusion injury areassociated with the production of alarmins. It has been described, that polymorphisms within the tlr4 gene are associated with reduced responses to alarmins. Aims: In this study, the association of polymorphism (rs4986790) Asp299Gly and (rs4986791) Thr399Il) tlr4 gene and tlr4 expressionon peripheral monocytes were examined, in patients with kidney transplant and healthy controls. The relationship of tested parameters in transplanted kidney patients with kidney function and morbidity in those patients were examined. Type of study: A cross-sectionalstudy. Material and Methods: Isolation of DNA was done by commercial colonies for isolation G-spinTM Genomic DNA Extraction Kit (Intron Biotechnology, Seul, South Corea). Polymorphisms were determined by real time PCR. Among the data collected from the patient groups which related to the function ofthe transplanted kidneys were: datadonors, recipients, HLA matching, medications, duration of coldischemia and complications. SPSS 16.0 was used for statistical analysis, p<0.05 was considered significant. Results: There was no differencein the distribution of wild and mutant alleles between the two groups of subjects. Asp299Gly polymorphism was present in 14.3% of the transplanted patients, while the Thr399Ile was in 11.3%. The expression of tlr4 and CD14 on peripheral blood monocytes were analysed by flow cytometry. Significantly lower percentages of monocytes (6.4%) (MannWhitneytest,p =0.001) were observed in the control group than within the kidney transplant recipients (7.3%). Expression of tlr4 and CD14 on peripheral monocytes were significantly higher in transplant patients. Patients treated with tacrolimus had significantly higher levels of tlr4 MFI. In the group of patients transplanted with AA-wild-type for Asp299Gly(rs4986790) there was a significantly higher expression of tlr4 (MannWhitney test,p =0.004), as well as, for the CCwild type for Thr399Ile(rs4986791), whereit was 107.1 (interquartile range 96.2 to 119.9) (MannWhitney test, p=0.008). Time elapsed since transplantation had no effect on tlr4 expression. We examined the effect of genotype on biochemical hematological parameters in patients with kidney transplant. Lower levels of red blood cells (Kruskal Wallis test, p=0.037), creatinine clearance (Kruskal Wallis test, p=0.019) and hemoglobin (Kruskal Wallis test, p=0.009) were observed in the AAwild-type rs4984790, while the highest measured glucose was among the AG heterozygote group. Urea values were significantly higher in AA-wild type. In the CC wild type of rs4986791 had significantly the lowest values of erythrocytes (Kruskal Wallistest, p=0.027), creatinine clearance (Kruskal Wallistest, p =0.030) and hemoglobin (Kruskal Wallis test, p=0.049). The median duration of cold ischemia time was 14.2 hours (range from 12.5 to 16.8 hours). The median duration of cold ischemia in hoursis in versely proportional to the creatinine clearance at the time of flow cytometry (Spearman's rank correlation coefficient ρ=-0.394, p=0.004). There was no association between polymorphisms and acute complications of kidney transplantation, such as delayed graft function or comorbidities after transplantation encountered. Conclusion: There was no difference in the distribution of genotypes among groups. Transplant patients have a high erexpression of tlr4 on peripheral monocytes. Genotype influences on the expression of tlr4 effect the mutant alleles in a way by causing a weaker expression of tlr4 on monocytes. Additionally, mutated allele carriers have better renal function, as measured by the creatinine clearance at the time of flow cytometry suggesting a possible protective role of mutant alleles. |
