الفهرس 
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Abstract
Diabetic nephropathy is one of the serious, chronic microangiopathic complications of type 2 diabetes (T2DM) and has become the most frequent cause of end-stage renal disease. It is characterized by the presence of albuminuria and/or proteinuria in a diabetic patient, arterial blood pressure elevation, decline in glomerular filtration rate (GFR), and high risk of cardiovascular morbidity and mortality.
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that play pivotal roles in the regulation of a very large number of biological processes, such as cell proliferation, cell differentiation, cell death, and inflammatory responses. PPARs include three known subtypes PPAR α, PPAR γ, and PPARβ/δ. It plays many functional roles in different organs and tissues; adipocyte differentiation, lipid and glucose homeostasis, and insulin sensitivity Two common polymorphisms of the PPARγ gene have been identified; a C/G transition in exon B results in a proline to alanine exchange at amino acid position 12; Pro12Ala. The other polymorphism is a C/T transition at position 161 in exon 6 C161T.
The present study aimed to test the association of thePPARγ2gene polymorphisms Pro12Ala and C161T in Egyptian patients with type 2 diabetic nephropathy, and illustrating the relationship (if any) in development of diabetic nephropathy. The sample study included one hundred and forty seven individual divided into three groups, 52 apparently healthy controls, 47 type 2 diabetic patients without nephropathy, and 48 type 2 diabetic patients with nephropathy. To all the studied groups, fasting serum (FSG), postprandial (PP) glucose and glycated hemoglobin (HbA1c), total cholesterol, HDL, LDL, TG,Creatinine, Uric acid were measured. eGFR and BMI, were calculated. Screening for PPARγ2Pro12Ala and C161T polymorphisms was done using polymerase chain reaction and restriction fragment length polymorphism techniques (PCR-RFLP).The frequency distribution of the two polymorphisms in all studied subgroups were in agreement with the Hardy-Weinberg (H.W.) equilibrium.
As regards to the frequency distribution ofPPARγ2Pro12Ala in controls in our study; it was 83.7%in Pro allele, and 16.3% for Ala allele. As for C161T frequency distribution in controls; it was 74% for ”C’ allele, and 26% for ”T” allele.
First, we comparedthe genotype frequency distribution ofPPARγ2Pro12Alabetween the control group (n=52) and the whole diabetic group (n=95) there were significant difference among both groups (p=0.018). This significance was more evident when combined Ala/Ala and Pro/Ala (Ala allele carriers) then compared to Pro/Pro (non carriers), the Pro/Pro genotype was significantly more frequent in diabetics when compared to nondiabetic (p=0.005).The Pro allele was associated with increased risk of susceptibility to T2DM with (OR=3.520, 95%CI=1.40-8.84,p=0.002). Furthermore when adjusted according to BMI;it showed almost the same range(OR:3.04, 95%CI=1.21-7.67,p=0.018).
The substitution from proline to alanine at codon 12 in PPARγ2Pro12Alapolymorphism has been found to modulate transcriptional activity. This substitution is close to the NH2-terminus of the protein in the ligand-independent activation domain, the activity of which is potentiated through phosphorylation by insulin. The structure and consequently the function of the protein may be affected by this amino acid change, since alanine favors the formation of a-helices while proline prevents it. The alanine isoform leads to the less efficient stimulation of PPARγ target genes and predisposes people to lower levels of adipose tissue mass accumulation, whichin turn may improve insulin sensitivity thus supporting the protective effect observed for Ala12 carriers, since decreased insulin sensitivity plays a central role in the pathogenesis of T2DM.
As regards forPPARγ2C161Tgenotypefrequency distributions the CC genotype was higher among controls: 57.7%,while in diabetics it was 42.1%.The TT frequency distribution showed lower distribution among controls 9.6%, while in diabetics showed higher level 13.7%,but this did not reach the level of statistical significance.
Second, we divided the diabetics, according to the state of nephropathy; we observed a significant association in PPARγ2Pro12Ala polymorphism with diabetic nephropathy. The ”Ala” allele was associated with risk reduction with OR:0.165,95% CI: (0.04-0.58) , while the ”Pro” allele was associated with increased risk of diabetic nephropathy with OR: 6.057, 95% CI:(1.7-21.39), p=0.002,and when adjusted according to hypertension and IHD ,OR was:4.69, 95% CI:(1.039-21.16),p=0.044.
PPARγ agonists (Thiazolidinedione for example rosiglitazone) are able to improve or attenuate the effects of kidney injury, including prevention of mesangial expansion, glomerulosclerosis, tubulointerstitial inflammation and fibrosis, tubular dilation and atrophy, arteriolar thickening, and diminish hyperlipidemia.This renoprotective action by PPARγ agonists is at least partially mediated via downregulation of renal disintegrin and metalloprotease-17 and angiotensin converting enzyme-2 shedding. Also, it is to be proved is a negative regulator of transforming growth factor-b1 (TGFb1)-induced fibrotic events, which have a great value, since fibrotic processes are important characteristics of renal dysfunction and renal diseases.Also, their role as antioxidant anti-inflammatory, antihypertensive, improving insulin sensitivity and antiatherosclerogenic compounds is evident.
As for genotype frequency distribution of PPARγ2C161Twith diabetic nephropathy in our study the ”C” allele was highest level among controls: 74%, while in diabetics without nephropathy it was 63.8%, and in diabetic with nephropathy it was 64.6%; however there was no statistically significance difference among groups.
Third,we divided the diabetics according to state of retinopathy. There were a significant association between PPARγ2Pro12Ala and diabetic retinopathy (DR), The Pro allele was 94% in diabetic retinopathy group while Ala allele was 6%in the control group (OR: 3.087, 95% CI :( 1.01-8.76), p=.007) (table:13a),this significance was lost when OR was adjusted for hypertension and IHD (OR:1.63, 95% CI (0.44-6.01), p=0.464).
As for PPARγ2C161T polymorphism our study showed a significant association with diabetic retinopathy, as there were a significant statistically difference between” C” allele and ”T” allele; in the control group and diabetics with retinopathy (OR:0.405, 95% CI:(0.22-0.75), p=0.003), when adjusted according to hypertensionand IHD; OR was: 0.3, 95% CI:(0.013-0.72),p=0.007), also between diabetics with retinopathy and without retinopathy (p=0.006). To our knowledge, this is one of the first studies exploring the association of PPARγ2C161T and diabetic retinopathy.
Fourth, we analyzed the relationship betweenPPARγ2Pro12Alaand C161T polymorphisms and markers of kidney function, glycemic control and BMI. In regards for HbA1c level in diabetics; the Ala allele carrier had lower median of HbA1c 7.4%(5.7-10.2), than the Pro/Pro genotype:8.55%(5.3-12.4), but it did not reach level of statistical significance. Then we classified diabetic group according to a cut-off value(7.5%) which has a more clinical relevance, there was a significant association among the different gene types noted p=0.045.
In the present study Albumin/creatinine ratio showed a significant higher level between Pro/Pro genotype versus and Pro/Ala and Ala/Alagenotypes among diabetics.
PPARγ2 is a lipid –activated transcription factor with a key role in the expression of genes involved in adipocyte differentiation and function. In the current work, we classified the studied population according to their BMI and assessed the relation between the studied SNPs and different BMI classes. A significant difference was noted in genotype distribution of PPARγ2 Pro12Ala in relation to different BMI classes (p=0.044) in the control group, and in particular when comparing those with Pro/Ala to those with Pro/Pro genotype (p=0.009). But this association was not significant in the total studied sample or the diabetics.
Lastly, to evaluate the interaction between PPARγ2Pro12Ala and C161T polymorphisms, we divided diabetic subjects into four groups according to the combination of the two genotypes from each SNP, and compared the studied parameters among the groups; group I: Pro/Pro with C/C genotype (n=38), group II: Pro/Pro with a T allele (n=48),group III: C/C with an Ala allele (n=3),group IV: T allele with an Ala allele (n=6).
As for the association of PPARγ2Pro12Ala and C161T with lipid profile; our study showed a significant difference in triglycerides among the different combination groups of the polymorphisms.
This gene is a confirmed T2DM susceptibility locus and is now one of almost 20 type 2 diabetes susceptibility loci identified over the last few years with other T2DM susceptibility loci. Research on how best to use this information is under continuous study, given the possible heterogeneity in the magnitude of the association among populations and with environmental factors, a more systematic assessment locus should also be used as a genetic factor with which to investigate both gene-gene and gene environment interactions with type 2 diabetes. Lastly, even though there were variations due to genetic factors and we understand how a drug works in the body,pharmacological intervention needs special attention in that it creates supraphysiological status, which may wipe out the moderate effect of genetic variation or act in a different way.
from the present study the following could be concluded:
1. The PPARγ2Pro12Ala polymorphism was significantly associated with risk of T2DM and diabetic nephropathy.
2. The PPARγ2C161T polymorphism showed no association with the development of diabetic nephropathy among the studied sample of Egyptian population however it was significantly associated with diabetic retinopathy.