الفهرس 
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Abstract
5. Summary
Type 2 diabetes is a worldwide chronic disease associated with several life-threatening complications and the protection from progression of renal injury in daibetic patient remains a challenge. Risk factors for nephropathy were found to be poor glycemic control, long duration of diabetes, hypertension and microalbuminuria. There is an urgent need to launch a novel early diagnostic markers targeting primary mechanisms contributing to renal damage to tackle the potential pathogenic complexity of diabetic nephropathy, and for future treatment of this complication.
Inflammatory cytokines are implicated in diabetic nephropathy, from development of the initial stages of diabetes to progression and to late stages of renal failure. The recognition of these molecules as significant pathogenic factors in this complication will provide new diagnositic markers.
Examining changes in the expression of pathogenic genes involved in inflammatory pathways will uncover important information regarding the pathogenic mechanisms of diabetic nephropathy. Likewise, this effort will also facilitate the identification of high-risk patients and perhaps lead to novel early diagnostic markers. In our study we used leukocytes as an accessible model to examine the potential role of our candidate genes as molecular markers for the DN progression.
The aim of this thesis was to explore the role of 4 candidates genes (IL-18, PPARG, THBS-1and TGF-β1) in the diagnosis and progression of DN and to explore the effets of T2DM on the expression levels of these genes.
In this study, a total of 55 blood samples from healthy controls (n=15), T2DM (n=20, WHO criteria), and DN (n=20, diagnosed based on ADA 2014). Full history including age, sex, BMI, diabetic duration, SBP, DBP were recorded for all subjects. All subjects of study were subjected to clinical examination as well as laboratory investigations including diabetic profile, lipid profile, kidney profile inflammatory markers and albuminurea.
Blood samples from all subjects of study were collected and RNA was extracted from leukocytes using TRiazol reagent then the extracted RNA was reverse-transcribed to cDNA, and used as templates in qPCR reactions. Data were analyzed using SPSS software. Significance was determined statistically at P-value ˂0.05. Data were expressed as mean ± SEM. IL-18 and PPARG showed statistically significant increases by comparing DN group relative to T2DM group. All DN patients exhibited significant increase urinary albumin when compared to both T2DM and control groups. Although both TGF-β 1 and THBS-1 showed differential expression within T2DM and DN groups.
To examine whether leukocyte gene expression is associated with clinical characteristics of the patients, correlation analysis was performed. A significant positive correlation was found between relative IL-18 expression and both FBG and HbA1c levels in DN group. PPARG was positively correlated with VLDLc level in DN group. In addition, relative PPARG expression in T2DM patients was positively correlated with CRP level, THBS-1 expression and TGF-β1 mRNA expression. There was a significant positive correlation between THBS-1 expression and ACR in DN group. TGFβ-1 mRNA expression level was positively correlated with THBS-1 in both DN and T2DM groups. In addition, relative TGFβ-1 expression level was positively correlated with ACR in DN group. from these data we can conclude the strength of the relationship between our candidate genes and the glycemic control of patients.
A ROC curve was generated to examine the diagnostic value (sensitivity and specificity) of using the relative expression of our candidate genes in WBCs as binary classifier diagnostic test. According to (Sox et al. 1989) an AUC ≥ 0.7 indicates the acceptance of a marker in diagnosis. The areas under the ROC curve (AUC) for IL-18 PPARG, THBS-1 and TGF-β1 were (0.975, 0.881, 0.629, and 0.615, respectively) indicating the probability of using both IL-18 and PPARG as markers in diagnosis of nephropathy population. On the other hand, the relative expression of both TGF-β1 and THBS-1 in WBCs are not recommended as a binary classifier between DN and T2DM patients (AUC˂0.7).
A follow up of biochemical parameters for the patients and control individuals were examined one year after initial evaluation. The changes in values of FBG, 2h PPBG, HbA1c, WBCs, CRP, TC, HDLc and VLDLc showed no significant difference from the previously collected data within each group but still higher than results of control group. In contrast, there was a significant increase from the initial values in ACR, urea, creatine, LDLc and TGs within DN group but are almost steady in T2DM group. Interestingly, about 47% of the patients in DN group developed change in ACR level, shifting from microalbuminuria to macroalbuminuria. Serum albumin was significantly decreased within DN group but was within the normal range in T2DM group.
To assess the prognostic potential of our candidate genes, correlation analysis was performed between the biochemical follow up data and the determined initial data of gene expression values. Within the DN group, there was a significant positive correlation between IL-18 relative gene expression and ACR. A highly significant correlation between PPARG gene expression in leukocytes and LDLc values was noted. There was a significant correlation between TGF-β1 relative expression and FBG level.
In conclusion, IL-18 and PPARG gene analysis of expression in WBCs of T2DM and DN patients demonstrated that mRNA levels of IL-18 and PPARG genes might be a valuable biomarkers of identifying nephropathy in patients with diabetes and might be a mirror for changes in kidney function indicating an important pathway in the pathogenesis and development of DN. Our results also revealed that both THBS-1 and TGF-β1 have elevated expression levels in T2DM and DN but cannot be a differentiating marker for nephropathy progression in diabetic patients.
It is recommended to be confirmed in a larger group of patients and long term follow up is recommended in order to accurately detect the correlation between DN progression and gene expression.