Andrzej Ksiazek

Toll-like receptor 4 gene polymorphism and early onset of diabetic retinopathy in patients with type 2 diabetes

Toll-like receptor 4 (TLR4) is an important mediator of innate immunity. Type 2 diabetes (DM2) might be associated with changed innate immune response. We investigated whether the polymorphisms in the TLR4 gene are associated with diabetic retinopathy (DR). The study group of 864 patients with DM2 and 420 healthy individuals were genotyped. In the patient group 352 subjects were diagnosed with DR. Out of the remaining 512, 140 had DM2 for > or = 10 years but no DR. In the DM2 group 7.4% of patients were heterozygous for the Asp299Gly polymorphism compared with 6.5% controls. For Thr399Ile polymorphism there were 7.2% heterozygotes vs 6.2% controls. In most cases, the linkage disequilibrium between the minor alleles Gly299 and Ile399 was confirmed. Increased frequency of both heterozygous genotypes was observed in patients with retinopathy (11.2% for the Asp299Gly). The frequency of the G allele was significantly higher in patients with early onset retinopathy (n = 80) vs patients without DR (odds ratio = 5.0, and 95% confidence interval = 2.33-10.71). In contrast, in the entire retinopathy group, the odds ratio for the G allele was 1.88 (95% confidence interval = 0.93-3.79). In the multivariate logistic regression analysis, the G allele of Asp299Gly was an independent risk factor of early onset DR (p < 0.001). In conclusion, our results suggest an association between the Asp299Gly polymorphism of the TLR4 gene and early onset of DR in the DM2 patients. Thus the G allele may be a predictor of increased risk of retinopathy.

Polymorphism of the renalase gene in end-stage renal disease patients affected by hypertension

BACKGROUND Renalase is a novel flavin adenine dinucleotide-dependent amine oxidase that is secreted by the kidney. It circulates in the blood and modulates the cardiac function and systemic blood pressure. Insufficiency of renalase in patients with chronic kidney disease may explain the frequent occurrence of hypertension among patients with end-stage renal disease (ESRD) and an increased risk of cardiovascular events in this group. The aim of the study was to assess the relationship of two renalase gene polymorphisms with hypertension in dialysed patients. METHODS Rs2576178 polymorphism was genotyped in 369 patients, rs10887800 polymorphism was genotyped in 421 dialysed patients, using polymerase chain reaction (PCR) and subsequent cleavage with Msp I and Pst I restriction endonucleases. RESULTS Genotype distribution and allele frequencies of rs2576178 polymorphism were compared in the following subgroups of patients: dialysed patients with hypertension: ESRD HY + (n = 200) and dialysed patients without hypertension: ESRD HY - (n = 169). There was a significant difference in the frequency of the G allele carriers. G allele carriers were associated with a 1.55 times higher risk of hypertension [odds ratio (OR) = 1.55; 95% confidence interval (CI): 1.023-2.357, P = 0.039]. Distribution of genotypes and frequencies of alleles of rs10887800 polymorphism were compared in the following subgroups of patients: ESRD HY + (n = 278) and ESRD HY - (n = 143). The G allele carriers were recognized with a significantly higher frequency in ESRD HY + patients (0.46 in ESRD HY + versus 0.37 in ESRD HY - ) [OR = 1.76; 95% CI: (1.159-2.667, P = 0.008)]. CONCLUSIONS Our results are the first to suggest an association between renalase gene polymorphisms analysed and hypertension in dialysed patients. It may be an important step towards gaining a deeper insight into cardiovascular pathophysiology. Furthermore, it might provide an optimal treatment and better prognosis for patients with chronic kidney disease.

Angiotensin II Type 1 Receptor Gene Polymorphism in End-Stage Renal Disease

The genes of the renin-angiotensin system (RAS) are involved in progression of renal failure. We examined the A1166C polymorphism at the angiotensin II type 1 (AT1R) locus in patients with end-stage renal disease (ESRD). The distribution of genotype and allele frequencies was compared in 430 dialysis patients and 260 healthy controls. DNA samples were amplified by the polymerase chain reaction (PCR) and amplification products were digested with BsuRI restriction enzyme. In the presence of cytosine (C) there is a restriction site for this enzyme, giving a fragment of 231 bp (C allele), whereas undigested 255 bp fragment indicates the presence of the A allele. The higher frequency of combined AC and CC genotypes was observed in the patient group than in controls (48.6 vs. 39.5%, p < 0.05). The average time from the onset of renal disease to ESRD in patients with C allele was significantly shorter than in those with AA genotype (6.3 vs. 13 years, p < 0.01). Positive family history of renal disease in patients with AC/CC genotype seems to increase this effect. Our results indicate that the presence of C allele of the AT1R locus polymorphism might be associated with faster deterioration of renal function.

Monocyte chemoattractant protein-1 (MCP-1) gene polymorphism as a potential risk factor for cardiovascular disease in hemodialyzed patients

AIM Polymorphism in the monocyte chemoattractant protein-1 (MCP-1) gene (A-2518G) has been associated with functional effects. The aim of the present study was to assess the effect of this polymorphism on end-stage renal disease (ESRD) and cardiovascular disease (CVD) in hemodialyzed patients. METHODS A total of 720 patients with ESRD treated with hemodialysis (450 patients with CVD) and 325 healthy control subjects were genotyped for the MCP-1 -2518 polymorphism by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure. RESULTS There was a significant difference in genotype frequencies between entire group of hemodialyzed patients and controls (p<0.01). The odds ratio for the risk allele was 1.85, 95% CI 1.49-2.32 (p<0.01). Hemodialyzed patients were divided into subgroups with CVD (n=450) and without CVD (n=270). The G allele carriers occurred with significantly higher frequency in patients with CVD (62% vs. 38% in patients without CVD and 36% in controls). The odds ratio for the risk allele for patients with CVD vs. those without CVD was 2.17, 95% CI 1.71-2.79. There was no statistically significant difference in the distribution of MCP-1 genotypes between ESRD patients without CVD and healthy controls. CONCLUSION Our results demonstrate for the first time an association between the polymorphism in the regulatory region of the MCP-1 gene and susceptibility to CVD in hemodialyzed patients.

Transcription factor 7-like 2 (TCF7L2) gene polymorphism and complication/comorbidity profile in type 2 diabetes patients

Transcription factor 7-like 2 gene (TCF7L2) has been associated with type 2 diabetes. We investigated the association of the rs7903146 SNP in this gene with clinical profile of type 2 diabetes (T2DM) patients. The study involved 980 patients with diabetic nephropathy (44%), diabetic retinopathy (42%), CVD (65%) and early onset of diabetes (45%) and 924 healthy controls. Subjects were genotyped for rs7903146 by PCR-RFLP. Genotype frequencies significantly differed between T2DM patients and controls (p<0.01, odds ratio (OR) for TT genotype 2.49 (95% confidence interval (CI) 1.84-3.39). An association was observed between rs7903146 and nephropathy (p<0.001), with 22% of TT homozygotes in this subgroup vs. 11% in patients without nephropathy (p=0.006, OR for TT 2.83, 95% CI 1.94-4.13). Association was stronger in patients with early onset of diabetes (34% of TT vs. 12% in the late onset, p<0.001). In DN group 71% of TT homozygotes had an early onset (OR 7.64, 95% CI 4.98-11.73 vs. controls). Our results confirm association of rs7903146 in the TCF7L2 gene with increased risk of type 2 diabetes. The T allele is strongly associated with nephropathy, especially in early onset of diabetes.

TGF-β1 and TSC-22 Gene Polymorphisms and Susceptibility to Microvascular Complications in Type 2 Diabetes

Background/Aim: There is a strong evidence for the involvement of genetic factors in diabetic microvascular complications. The aim of our study was to investigate the role of molecular variants of the TGF-β1 (transforming growth factor beta 1) and the TSC-22 (transforming growth factor beta stimulated clone 22) genes in diabetic nephropathy and diabetic retinopathy in type 2 diabetes. Methods: A case-control study was conducted in 503 patients and 400 healthy subjects. DNA samples were genotyped by polymerase chain reaction and restriction fragment length polymorphism methods. Results: Among the patients, 245 had diabetic nephropathy, 195 had retinopathy, and 168 were free from complications. All subjects were genotyped for T869C and C –509T polymorphisms of the TGF-β1 gene and for –396 polymorphism of the TSC-22 gene. A significantly increased frequency of the CC genotype of the T869C polymorphism was observed in patients with nephropathy and retinopathy (33 and 48%, respectively, vs. 19 and 15%, respectively, in controls and patients free from complications). The frequency of the C allele was also higher (0.58 for nephropathy and 0.64 for retinopathy vs. 0.42 in controls). The G allele of the TSC-22 polymorphism was associated with an increased risk of diabetic nephropathy (frequency 0.15 vs. 0.07 and 0.06, respectively, in patients free from complications and controls). An interaction was observed between the G allele of the TSC-22 polymorphism and the C-allele of the TGF-β polymorphism. Conclusions: Our data suggest the association of TGF-β T869C gene polymorphism with an increased risk of nephropathy and retinopathy in type 2 diabetes patients. It interacts with the TSC-22 gene involved in the TGF-β signaling pathway, promoting the development of diabetic nephropathy.

Heat-shock protein gene polymorphisms and the risk of nephropathy in patients with Type 2 diabetes

HSPs (heat-shock proteins) are molecular chaperones synthesized under stress conditions, and are involved in renal cell survival and matrix remodelling in acute and chronic renal diseases. In the present study, we investigated whether the HSP70 gene polymorphisms affect susceptibility to DN (diabetic nephropathy) in patients with T2DM (Type 2 diabetes mellitus). The study group consisted of 452 patients with nephropathy. Two control subgroups involved 340 healthy individuals and 132 patients with T2DM lasting > or =10 years who were free of nephropathy. Subjects were genotyped for the HSP70-1 +190 G/C and -110 A/C, HSP70-2 +1267 A/G and HSP70-hom +2437 T/C polymorphisms by PCR, followed by digestion with restriction endonucleases. There were no statistically significant differences in genotype distribution between patients with T2DM with DN and controls for the HSP70-hom polymorphism. Significant differences were observed for HSP70-1 and HSP70-2 polymorphisms. CC homozygotes of the -110 and +190 HSP70-1 polymorphisms were more frequent in patients with T2DM with DN compared with healthy controls (22 compared with 6% and 15 compared with 6.5% respectively; P<0.01). The OR (odds ratio) for the risk allele was 2.17 [95% CI (confidence interval), 1.73-2.72] for the -110 A/C and 1.74 (95% CI, 1.40-2.15) for +190 G/C polymorphisms. A strong association with DN was found for the +1267 HSP70-2 polymorphism. The GG genotype and the G allele were associated with DN, with the OR for the G allele being 4.77 (95% CI, 3.81-5.96). All GG homozygotes in the patient group had higher LDL (low-density lipoprotein)-cholesterol levels than AA homozygotes (P<0.01), suggesting that the observed effect might be associated with this cardiovascular risk factor. These patients progressed faster to end-stage renal failure than those with other genotypes. In conclusion, our results indicate that the HSP70-1 and HSP70-2 polymorphisms are associated with renal complications in T2DM and may be useful in identifying patients with increased risk of DN.

Monocyte chemoattractant protein (MCP-1) A-2518G gene polymorphism in stroke patients with different comorbidities

OBJECTIVES The aim of our study was to assess the effect of A-2518G polymorphism in the monocyte chemoattractant protein-1 gene on development of stroke. DESIGN AND METHODS A total of 194 patients with stroke and 320 healthy controls were genotyped for the MCP-1 gene -2518 polymorphism. RESULTS There was a significant difference in genotype frequencies between ischemic stroke patients and controls (p=0.01). Stroke patients were subdivided according to gender, presence of renal disease, small-vessel disease, diabetes, atherosclerosis and hyperlipidemia. There were differences in genotype frequencies between stroke patients with atherosclerosis and controls (p=0.03), and in allele frequencies between diabetic patients and controls (p=0.04). In hyperlipidemia, the OR 2.33 for the GG genotype may be due to stroke, because it was found only vs. controls and not vs. group without hyperlipidemia. CONCLUSIONS Our results demonstrate an association between the polymorphism in the regulatory region of MCP-1 gene and susceptibility to ischemic stroke.

Phosphate, urea and creatinine clearances: haemodialysis adequacy assessed by weekly monitoring

BACKGROUND The specific distribution of phosphate and the control mechanisms for its plasma level makes phosphate kinetics during haemodialysis (HD) considerably different from those of urea and creatinine and makes the quantitative evaluation of adequacy of phosphate removal difficult. We propose the application of equivalent continuous clearance (ECC) as a phosphate adequacy parameter and compare it with ECC for creatinine and urea. METHODS Three consecutive dialysis sessions were evaluated for 25 patients on maintenance HD. Concentrations of phosphate, urea and creatinine in plasma were measured every 1h during the treatment and 45 min after, and every 30 min in dialysate. ECC was calculated using the removed solute mass assessed in dialysate and weekly solute profile in plasma. Similar calculations were performed also for the midweek dialysis session only. Different versions of the reference concentration for ECC were applied. RESULTS ECC with peak average reference concentration was 5.4 ± 1.0 for phosphate, 7.0 ± 1.0 for urea and 4.7 ± 1.0 mL/min for creatinine. ECC for urea and creatinine were well correlated in contrast to the correlations of ECC for phosphate versus urea and creatinine. Midweek ECC were higher than weekly ECC, but they were well correlated for urea and creatinine, but only weakly for phosphate. CONCLUSIONS HD adequacy monitoring for phosphate may be performed using ECC, but it is less predictable than similar indices for urea and creatinine. The values of ECC for phosphate are within the range expected for its molecular size compared with those for urea and creatinine.

Common polymorphism in the cannabinoid type 1 receptor gene (CNR1) is associated with microvascular complications in type 2 diabetes

Endocannabinoids exert their biological effects via interaction with G-protein coupled cannabinoid receptors CB1 and CB2. Polymorphisms in the CNR1 gene (encoding CB1 receptor) were previously found to be associated with dyslipidemia and cardiovascular diseases. We investigated a role of the polymorphism in CNR1 gene in type 2 diabetes and its complications. The study involved 667 T2DM patients and 450 healthy individuals. All subjects were genotyped for G1359A polymorphism by PCR-RFLP procedure. Genotype frequencies did not differ significantly between patients and controls. The statistically significant differences were seen between T2DM patients with diabetic nephropathy (DN) and those without it (OR for risk allele 2.84, 95% CI 2.04-3.94, p<0.0001). There were also differences between patients with diabetic retinopathy (DR) and those without DR (OR for risk allele 1.81, 95% CI 1.30-2.53, p=0.0005). No differences were observed in diabetic neuropathy. The A allele was more frequent in patients with coexisting cardiovascular disease (CVD) compared to patients without CVD (p=0.0044). The novel finding of our study is the association of the G1359A polymorphism with diabetic nephropathy and diabetic retinopathy in patients with T2DM. This polymorphism was also associated with cardiovascular disease in the patient group.