|Year : 2014 | Volume
| Issue : 3 | Page : 582-588
Study of the heat shock protein 70-1 gene polymorphism and the risk of nephropathy in type II diabetic patients
Naglaa M Ghanayem1, Maathir K El-Shafie1, Eman A.F. Badr1, Elsayed Elnour1, Said S Khamis2, Eman M Abd El Gayed Msc 1
1 Department of Medical Biochemistry, Faculty of Medicine, Menofia University, Menofia, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Menofia University, Menofia, Egypt
|Date of Web Publication||26-Nov-2014|
Eman M Abd El Gayed
Department of Medical Biochemistry, Faculty of Medicine, Menofia University, Shebein Elkom, Menofia
Source of Support: None, Conflict of Interest: None
To study whether the heat shock protein 70-1 (HSP70-1) gene polymorphism affects susceptibility to diabetic nephropathy (DN) in patients with type 2 diabetes mellitus (T2DM).
Heat shock proteins (HSPs) are molecular chaperones synthesized under stressful conditions and are involved in renal cell survival and matrix remodeling in acute and chronic renal diseases.
Patients and methods
This study was carried out on 80 patients divided into three groups: 30 patients with T2DM with DN (group I), 30 T2DM patients without DN (group II), with duration of diabetes more than 10 years in both patient groups, and 20 healthy individuals who served as controls (group III). All studied participants were subjected to a full assessment of history, general clinical examination, and laboratory investigations including determination of fasting blood glucose, total cholesterol, glycated hemoglobin (HBA1c), serum urea, serum creatinine, and urinary albumin to creatinine ratio. The HSP70-1 gene polymorphism -110 A/C was determined using the PCR-restriction fragment length polymorphism technique.
The results of the present study showed a highly significant statistical difference between group I and group II in family history, systolic and diastolic blood pressure, and duration of diabetes. Significant differences were observed for the -110 A/C genotype distribution on comparing the three studied groups, with increased frequency of the CC genotype in diabetic patients with DN, increased frequency of the AC genotype in diabetic patients without DN, and increased AA genotype frequency in the controls. CC genotypes of -110 A/C might represent a genetic risk factor for DN on comparing group I with group III.
This result indicates that HSP70-1 CC genotypes of -110 A/C are highly associated with renal complications in patients with T2DM and can be a useful marker in identifying patients with an increased risk of DN.
Keywords: diabetic nephropathy, heat shock protein 70-1
|How to cite this article:|
Ghanayem NM, El-Shafie MK, Badr EA, Elnour E, Khamis SS, Abd El Gayed EM. Study of the heat shock protein 70-1 gene polymorphism and the risk of nephropathy in type II diabetic patients. Menoufia Med J 2014;27:582-8
|How to cite this URL:|
Ghanayem NM, El-Shafie MK, Badr EA, Elnour E, Khamis SS, Abd El Gayed EM. Study of the heat shock protein 70-1 gene polymorphism and the risk of nephropathy in type II diabetic patients. Menoufia Med J [serial online] 2014 [cited 2020 Feb 24];27:582-8. Available from: http://www.mmj.eg.net/text.asp?2014/27/3/582/145519
| Introduction|| |
Diabetes mellitus (DM) is a major public health problem and the prevalence of type 2 diabetes mellitus (T2DM) is increasing worldwide  . Diabetic microvascular complications are the major causes of morbidity and premature mortality in T2DM  . Diabetic nephropathy (DN) has become the leading cause of end-stage renal failure worldwide. There are multiple evidences implicating genetic factors in the susceptibility to DN and retinopathy  .
Heat shock proteins (HSPs) are molecular chaperones synthesized under stressful conditions. They are induced by denatured proteins during heat shock, ischemia, and other cellular stresses  . The major classes of HSPs play essential roles in the folding/unfolding of proteins  , the assembly of multiprotein complexes, the transport/sorting of proteins into correct sub cellular compartments, cell-cycle control, cell signaling mechanisms, and the protection of cells against stress/apoptosis  .
Oxidative stress plays an important role in renal diseases  . Previous studies have documented the crucial role of HSPs in renal cell survival and matrix remodeling in several acute and chronic renal diseases  . The heat shock protein 70 (HSP70) (70 kDa HSP) family is the most abundant in eukaryotic cells and is essential for cell survival under stressful conditions  . In humans, three genes encoding members of the HSP70 class are mapped within the major histocompatibility complex class III region (6p21.3): HSP70-1 (HSPA1A), HSP70-2 (HSPA1B), and HSP70-hom (HSPA1L). HSP70-1 and HSP70-2 encode an identical heat-inducible protein HSP70, but differ in their regulatory domains, whereas HSP70-hom encodes a non-heat-inducible form  . These genes are polymorphic, with some variants potentially accounting for a change in function and susceptibility to stress tolerance  . HSP70 gene polymorphisms were found to be risk factors in several human disorders  and they might play an important role in susceptibility to and/or progression of DN  .
| Aim|| |
The aim of this study is to study the association between HSP70-1 polymorphism at -110 A/C and susceptibility of patients with T2DM to develop DN.
| Participants and methods|| |
This study was carried out on 80 individuals: 60 diabetic patients and 20 healthy individuals who served as controls. They were selected from the Urology Unit, Internal Medicine Departments, Menofia University Hospitals. These participants were divided into three groups: group I included 30 patients with T2DM with DN, 13 men and 17 women, mean age 54.15 ± 6.09 years. The mean duration of diabetes in these patients was 16.20 ± 4.13 years. They were on maintenance dialysis (ESRD). DN was determined on the basis of questionnaires, medical files, and laboratory data. DN was diagnosed clinically when the patient had an albumin/creatinine ratio of more than 300 μg/mg in the absence of hematuria or infection. Group II included 30 T2DM patients without DN, 16 men and 14 women, mean age 54.7 ± 6.11 years. The mean duration of diabetes in these patients was 13.87 ± 2.71 years. These patients had albumin/creatinine ratio less than 30 μg/mg in two determinations.
Group III included 20 healthy volunteers, age and sex matched with the patients, with no history of diabetes, renal, or cardiovascular diseases. Their mean age was 57.60 ± 5.14 years. Diabetic patients with duration of diabetes less than 10 years or those who had microalbuminuria were excluded from the study. Written informed consent was obtained from all the participants in accordance with principles of the Ethical Committee of Menofia Faculty of Medicine.
All participants were subjected to the following
Full assessment of history, a thorough clinical examination, laboratory investigations including measurement of serum total cholesterol, serum urea and creatinine, urinary albumin/creatinine ratio, fasting blood glucose (FBG), glycated hemoglobin (HbA1c), and detection of HSP70-1 gene polymorphism -110 A/C by PCR/restriction fragment length polymorphism.
- Random urine samples were collected for determination of creatinine and microalbumin in urine to calculate the A/C ratio , .
- After 12 h overnight fasting, 9 ml of venous blood was withdrawn from every fasting participant by sterile vein-puncture and divided into three tubes. One milliliter of blood was transferred into an EDTA tube for quantitative colorimetric determination of HbA1c  .
- One ml of blood was transferred into a sodium fluoride tube and 2 ml blood was collected after 2 h for enzymatic colorimetric determination of blood glucose  .
- Two milliliter was transferred into a plain tube and allowed to clot at 37°C and centrifuged for 10 min at 4000 rpm. The clear supernatant serum was separated from the clot and kept frozen at -80°C until determination of serum urea  , serum creatinine  , and serum total cholesterol  .
The remaining five ml of blood was transferred into an EDTA-containing tube and used for lymphocyte separation for further molecular analysis (Bio test AG, Dreieich, Germany). DNA from lymphocyte samples was isolated using the QIAGEN extraction kit (Hilden, Germany). DNA eluted in buffer AE was stored at −20°C for further PCR procedures.
PCR for the HSP70-1 gene was carried out to a total volume of 25 μl containing 10 μl genomic DNA, 1 μl of each primer, 2.5 μl of 10× Taq polymerase buffer, 2 μl of 1.5 mmol/l MgCl 2 , 0.5 μl of AmpliTaq DNA polymerase (Genecraft, Germany), 1.0 μl of dNTPs (Stratagene, USA), 2 ul of dimethyl sulfoxide, and 5 ul distal water  .
HSP70-1 was analyzed using the following primers (Midland, Texas, USA):
(1) Forward: 5΄-CGCCATGGAGACCAACACCC-3΄.
(2) Reverse: 5΄-GCGGTTCCCTGCTCTCTGTC-3΄.
PCR amplification for the HSP70-1 gene was performed separately in a programmable thermal cycler GeneAmp PCR System 2400 (PerkinElmer).
For the HSP70-1 gene: one cycle at 94°C for 5 min, followed by 40 cycles at 94°C for 1 min; 62°C for 1.5 min; 72°C for 2 min; and one final cycle of extension at 72°C for 10 min.
The amplification products were separated by electrophoresis through a 3% agarose gel stained with ethidium bromide.
For the HSP70-1 gene, one band was observed at 488 bp ([Figure 1] and [Figure 2]).
|Figure 1: The heat shock protein 70-1 gene; lanes from 2-12 show the length of the PCR amplicon, which is 488 bp ladder of 100 bp (lane 1).|
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|Figure 2: The heat shock protein 70-1 gene -110 A/C polymorphism; the uncut fragment was 488 bp and digestion products were 215, 201, and 72 bp. Lane 1 indicates ladder 100 bp. Lanes 6, 7, and 13 indicate the AA genotype (215, 201, and 72 bp). Lanes 4, 5, 9, 10, and 11 indicate the AC genotype (488, 215, 201, and 72 bp). Lanes 2, 3, 8, and 12 indicate the CC genotype (488 bp).|
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The HSP70-1 −110 A/C polymorphism using the restriction fragment length polymorphism technique
Ten microliter of the PCR products for the HSP70-1 were mixed with 2 μl (2 U) of FastDigest SacI restriction enzyme (provided by Fermentas) with 17 μl nuclease-free water and 1 μl of 10× FastDigest Buffer.
The mixture was mixed well and incubated for at 37°C for 30 min, and then 10 μl of the products were loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The uncut fragment was 488 bp and digestion products were 215, 201, and 72 bp.
Results were collected, tabulated, and statistically analyzed using an IBM personal computer and statistical package SPSS version 11. The χ2 -test was used to study the association between two qualitative variables. Odds ratio describes the probability that individuals who are exposed to a certain factor will have a disease compared with individuals who are not exposed to the factor. The Student t-test was used for comparison of quantitative variables between two groups. The ANOVA (F) test was used for comparison of quantitative variables between three or more groups. Multiple regression analysis was carried out to calculate the effects of risk factors as independent odds ratios with the effects of other confounders removed. P-value less than 0.05 was considered statistically significant.
| Results|| |
The study showed no statistically significant difference among the three studied groups in age and sex distribution ([Table 1]).There was a statistically significant increase in the duration of diabetes in group I compared with group II. There was a highly statistically significant difference in the prevalence of hypertension when both diabetic groups were compared. There was a highly statistically significant difference among the three studied groups in systolic blood pressure (SBP), diastolic blood pressure (DBP), and BMI. There was a high statistically significant increase in SBP and DBP in group I compared with group II, whereas there was a statistically significant increase in BMI. There was a high statistically significant increase in SBP, DBP, and BMI in group I compared with group III. There was a statistically significant increase in SBP and DBP in group II compared with group III, whereas there was a high statistically significant increase in BMI. There was a statistically significant difference between group I (diabetic with nephropathy) and group II (diabetic without nephropathy) in a family history of diabetes and a highly statistically significant difference when each diabetic group (with and without nephropathy) was compared with the control group ([Table 2]). There was a significant increase in FBG, 2 h postprandial, HbA1c, serum urea, serum creatinine, urinary albumin/creatinine ratio, and total cholesterol in group I when compared with groups II and III. On comparing group II with III, there was a significant increase in FBG, PPBG, serum creatinine, and serum total cholesterol, whereas a statistically nonsignificant difference existed in serum urea and urinary albumin/creatinine ratio ([Table 3]). There was a statistically significant difference in the genotype and allele distribution of the HSP70-1 (-110 A/C) polymorphism among the three studied groups. There was a statistically significant difference in the HSP70-1 (-110 A/C) polymorphism and allele distribution on comparing group I with group III and a statistically nonsignificant difference on comparing group I with group II, whereas on comparing group II with group III, there was a statistically nonsignificant difference in -110 A/C genotyping and a statistically significant difference in the HSP70-1 allelic distribution, with the highest frequency of the -110 AA polymorphism in the control group, AC polymorphism and A allele in group II, and an increased frequency of the CC polymorphism and C allele in group I ([Table 4]). The results showed that CC genotypes might be a genetic risk factor for DN when T2DM patients with nephropathy were compared with controls ([Table 5]). The results showed that CC genotypes are not genetic risk factors for DN when T2DM patients with DN were compared with those without DN ([Table 6]). The results showed that HbA1c, followed by sex, DBP, age, DM duration, and SBP are independent risk factors for DN ([Table 7]).
|Table 3 Statistical comparison of biochemical parameters of the groups studied |
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|Table 4: Genotype and allele distribution of the HSP70-1 (−110 A/C) polymorphism among the three groups studied |
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|Table 5: Association of the HSP70-1 (−110 A/C) polymorphism with diabetic nephropathy in group I versus group III |
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|Table 6: Association of the HSP70-1 (−110 A/C) polymorphism with diabetic nephropathy in group I versus group II |
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|Table 7: Multivariate logistic regression analysis of risk factors for DN |
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| Discussion|| |
DN is a major cause of end-stage renal disease, affecting 28.9% of new adult patients starting renal replacement therapy  . Previous studies have documented the crucial role of HSPs in renal cell survival and matrix remodeling in several acute and chronic renal diseases  . The present study showed that there was no statistically significant difference in age and sex distribution among the three groups studied. This is in agreement with Jeon et al.  , and Zahran et al.  and in contrast to Tanveen et al.  .
The present study showed that there was a statistically significant difference between the two diabetic groups (with and without DN) in the family history of diabetes and a highly statistically significant difference when each of the diabetic groups was compared with the control group. This is in agreement with Fava and Hattersley  , and not in agreement with Agarwal et al.  and Themeli et al.  . The present study showed that there was a statistically significant difference in the duration of diabetes on comparing diabetic groups (with and without nephropathy) with each other. This result is in agreement with that of Giorgino et al.  , who reported duration of diabetes as a risk factor for overt nephropathy and microalbuminuria.
In this study, there was a statistically significant difference among the diabetic patients and the control group in BMI. These results are not in agreement with those of Wu et al.  . Mohammed and Mitchell  reported that the association between obesity and insulin resistance is likely a cause-effect relationship.
In terms of the risk factors for DN, the present study found a highly statistically significant difference in the level of SBP and DBP on comparing the patient groups with each other and with the control group, with the highest level in patients with DN. These results are in agreement with many other researchers [25,28-33] who reported that this may be because of endothelial dysfunction associated with insulin resistance in patients with T2DM.
The present study showed a high statistically significant difference among the three groups studied in fasting and 2 h postprandial blood glucose. There was a statistically significant difference on comparing the two patient groups with each other and the control group. These results are in agreement with those of many other studies ,,,, that have reported that hyperglycemia has been shown to induce transforming growth factor β and VEGF production in the kidney, which are a common mediator of endothelial injury progression of all renal diseases.
This study showed a highly statistically significant difference in the levels of HbAlc% on comparing the three studied groups and a statistically significant increase in HbAlc in diabetics with nephropathy when compared with diabetics without nephropathy. There was also a statistically significant difference when each of diabetic groups was compared with the controls. These results are in agreement with those of Giorgino and colleagues [25, 32, 33, 39, 40], who found that the rate of formation of HbA1c is directly proportional to the concentration of glucose in the blood and that a decrease in HbA1c concentrations by 1% leads to an estimated reduction of 30% in the risk of microvascular complications in DM.
The present study found a highly statistically significant difference in the serum level of urea and creatinine on comparing patients with DN and those without DN and between diabetic patients with DN and controls. However, there was a statistically significant difference between diabetic patients without DN and control in serum creatinine and no statistically significant difference in serum urea. These results are in agreement with those of others ,,,,,, , who found that serum creatinine and BUN levels in T2DM patients were significantly higher when compared statistically with the controls. In contrast, Azar et al.  found no change in the kidney functions in normoalbuminuric diabetic patients.
The present study found a highly statistically significant difference in the urinary albumin/creatinine ratio on comparing patients with DN and those without DN and between diabetic patients with DN and controls, whereas there was no statistically significant difference on comparing patients without nephropathy and the control group. These results are in agreement with those of Ejuoghanran and colleagues , . This can be explained by the results of Brosius  , who reported that the early defect in autoregulation of renal perfusion makes it easier for albumin to leak from capillaries to renal glomerulus, thickening of the glomerular basement membrane and podocyte damage.
The present study found a highly statistically significant difference in the level of serum total cholesterol on comparing the three studied groups and two patient groups with each other and with a control group. These results are in agreement with those of [24, 49, 50, 52]. It was found that in T2DM, elevated serum total cholesterol is a risk factor for the development of DN. This can be because of increased plasma glucose concentration, which leads to increased glycosylation of proteins, particularly lipoproteins. Glycosylation of low-density lipoprotein has been shown to enhance its susceptibility to oxidation, which triggers the atherosclerotic processes  .
This study found a statistically significant difference in the distribution of the -110 A/C HSP70-1 polymorphism on comparing the three studied groups with increased frequency of the CC genotype and C allele in diabetic patients with DN, increased frequency of the AC genotype in diabetic patients without DN, and increased AA genotype and A allele frequency in controls. These results are in agreement with those of Buraczynska et al.  , who reported that significant differences were observed for the -110 A/C HSP70-1 polymorphism. CC homozygotes of the −110 polymorphisms were more frequent in diabetic patients with DN compared with healthy controls (22% compared with 6%).
In terms of the association of the HSP70-1 (-110 A/C) polymorphism and allele distribution with DN, this study found that CC genotypes and the C allele of the HSP70-1 (-110 A/C) polymorphism might be a risk factor for DN when T2DM patients were compared with controls. However, no association existed when T2DM patients were compared with those without DN. These results are in agreement with those of Buraczynska et al.  , who reported highly significant differences in the genotype distribution between diabetic patients with DN and healthy controls, and suggested that CC genotypes and the C allele of HSP70-1 (-110 A/C) might be genetic risk factors for DN. Furnrohr et al.  reported that HSPs play a role in the delivery and presentation of antigenic peptides and are considered to be involved in the pathogenesis of multi factorial diseases. Variations−110 A/C and +190G/C have been shown previously to be associated with Parkinson's disease Wu et al.  , celiac disease Ramos et al.  , and autoimmune thyroid disease Hunt et al.  .
The multivariate logistic regression analysis in this study showed that HbA1c, followed by sex, DBP, age, and DM duration are independent risk factors for DN. These results are in agreement with those of Buraczynska et al.  and Unnikkrishnan et al.  .
| Conclusion|| |
The study concluded that the HSP70-1 CC genotype of HSP70-1 (-110 A/C) is associated with susceptibility to DN in the T2DM patients studied. Poor glycemic control, hypertension, and longer duration of diabetes are independent risk factors for such complications.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]