| Abstract|| |
Heat-shock proteins (HSPs) are a group of proteins that function to protect cells and tissues against different types of damage. The aim of this work was to study the relationship between the genetic variation in HSP70 genes and the risk for development of nephropathy in Egyptian patients with Type 2 diabetes mellitus (DM). This study was carried out on 90 patients divided into three groups: 30 patients of Type 2 DM with nephropathy (Group I), 30 patients of Type 2 DM without nephropathy (Group II) with duration of diabetes > 10 years in both patient groups, and 30 healthy persons, who served as controls (Group III). All the studied patients were submitted to full history taking, complete clinical examination, and laboratory investigations including fasting blood glucose, glycated hemoglobin, renal function tests, and urinary albumin- to-creatinine ratio. HSP70-1 -110 AC, +190 G/C, HSP70-2 +1267 A/G, and shock protein70- hom +2437 T/C gene polymorphism were determined using the polymerase chain reaction- restriction fragment length polymorphism technique (PCR-RFLP). The results of the present study showed a highly statistically significant difference between Group I and Group II regarding family history, systolic and diastolic blood pressure, and duration of diabetes. There was a significant difference in the distribution of C allele of HSP70-1 -110A/C and +190 G/C and G allele of HSP70-2+1267A/G with more frequent detection in nephropathy group versus other groups, while there was no significant difference in genotype and allele distributions among the three studied groups for the HSP70-hom. It can be concluded that the C allele distribution of (HSP70-1 -110 A/C and HSP70+190 C/G) and the G allele distribution of HSP70-2 +1267A/G are associated with the susceptibility to renal complications in Egyptian patients with Type 2 DM.
|How to cite this article:|
Elshahed OM, Shaker OG. Heat Shock Protein 70 Gene Polymorphism in Egyptian Patients with Type 2 Diabetes Mellitus, with and without Nephropathy. Saudi J Kidney Dis Transpl 2020;31:787-95
|How to cite this URL:|
Elshahed OM, Shaker OG. Heat Shock Protein 70 Gene Polymorphism in Egyptian Patients with Type 2 Diabetes Mellitus, with and without Nephropathy. Saudi J Kidney Dis Transpl [serial online] 2020 [cited 2020 Sep 28];31:787-95. Available from: http://www.sjkdt.org/text.asp?2020/31/4/787/292312
| Introduction|| |
Diabetes mellitus (DM) is a major public health problem, and the prevalence of Type 2 DM is worldwide. Diabetic microvascular complications are the major causes of morbidity and premature mortality in Type 2 DM. Nephropathy has become the leading cause of end-stage renal failure worldwide. There are multiple evidences implicating genetic factors in the susceptibility to nephropathy and retino- pathy. Heat-shock proteins (HSPs) are mole cular chaperones synthesized under stressful conditions, and 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 multi- protein complexes, the transport/sorting of proteins into correct subcellular 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. In humans, three gene encoding members of the HSP70 class are mapped within the major histo- compatability complex. 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.,
The aim of this study is to evaluate the relationship between the genetic variations in HSP70-hom (+2437 T/C), HSP70-1 (-110 A/C and +190 G/C), and HSP70-2 (+1267 A/G) genes and the risk for nephropathy in patients with Type 2 DM.
| Subjects and Methods|| |
This study was carried out on 90 individuals: 60 diabetic patients and 30 healthy persons, who served as controls. They were selected from the Internal Medicine Departments, Cairo University Hospitals. These patients were divided into three groups:
- Group I: 30 patients with Type 2 DM with nephropathy. There were 13 males and 17 females, with a mean age of 54.11 ± 6.02 years. The mean duration of diabetes was 16.27 ±4.11 years. They were on maintenance dialysis for end-stage renal disease (ESRD)
- Group II: 30 patients of Type 2 DM without nephropathy. There were 16 males and 14 females, with a mean age 54.7 ± 6.11 years. The mean duration of diabetes was 13.87 ± 2.71 years.
- Group III: 30 healthy volunteers, age and gender matched with the patients, with no history of diabetes, renal, or cardiovascular diseases. Their mean age was 57.60 ±5.14 years.
A written consent was obtained from all study patients in accordance with principles of the Ethical Committee of Cairo Faculty of Medicine.
Diabetic patients with diabetes duration less than 10 years or had microalbuminuria diagnosed by microalbumin level > 30 mg/day, were excluded from the study.
All patients were submitted to the following: full history taking; thorough clinical examination; laboratory investigations including measurement of serum total cholesterol, serum urea and creatinine, albumin/creatinine ratio (ACR) in urine, fasting blood glucose (FBG), glycated hemoglobin (HbA1c), and genotyping of HSP70-hom (+2437 T/C), HSP70-1 (-110 A/C and +190 G/C) and HSP70-2 (+1267 A/G) polymorphisms by PCR-RFLP method.
| Statistical Analysis|| |
Laboratory results were collected, tabulated, and statistically analyzed by IBM personal computer and Statistical Package for Social Sciences (SPSS) version 11.0 (SPSS Inc., Chicago, IL, USA). Chi-square test and Fisher’s exact test were used to study association between two qualitative variables. Student’s (f) test was used for comparison between two groups having quantitative variables. ANOVA (τ) test was used for comparison between three or more groups having quantitative variables followed by post hoc test.
Multiple regression analysis calculated the effects of risk factors as independent odds ratios with the effects of other confounders. P < 0.05 was considered statistically significant.
| Results|| |
The demographic data of this study showed no difference between patients and controls [Table 1]. The results also showed a significant statistical association between the duration of diabetes and the prevalence of hypertension (P < 0.001) in Group I compared to Group II [Table 2]. There was a highly significant difference among the three studied groups regarding systolic blood pressure (SBP) (P < 0.001), diastolic blood pressure (DBP) (P < 0.001), and body mass index (BMI; P < 0.001) [Table 2]. There was a statistically significant difference between the three groups regarding the family history of diabetes (P < 0.001) [Table 2]. There was a highly statistically significant difference among the three studied groups regarding FBG (P < 0.001), postprandial blood glucose (PPBG) (P < 0.001), HbA1c (P < 0.001), serum urea (P < 0.001) and creatinine (P < 0.001), ACR (P < 0.001), and total cholesterol (P < 0.001) [Table 3]. There was no significant correlation between ACR and age, SBP, DBP, FBG, PPBG, HbA1c%, and cholesterol.
|Table 3: Statistical comparison of biochemical parameters among the three studied groups.|
Click here to view
HSP 70-1 -110 A/C polymorphism
The digestion products of Hsp70-1 gene 110A/C polymorphism were 215, 201, and 72 bp for AA and 488, 215, 201, and 72 bp for AC genotype and 488 bp for CC.
Regarding HSP 70-1 110 A/C genotypes, there was a significant increased frequency of the CC genotype and C allele of 110 A/C in Group I, AC in Group II, and AA in Group III compared to the other two groups. On comparing Group I with Group III, the results showed that, CC genotype of 110 A/C polymorphism increased the risk of nephropathy by 6.2 fold and AC genotype increased the risk by 6.0 fold and C allele increased the risk by 3.7 fold. On comparing Group I with Group II, the results showed that, CC genotype increased the risk of nephropathy by 2.9 fold and AC genotype increased the risk by 1.2 fold and C allele increased the risk by 0.9 fold [Table 4] and [Table 5].
|Table 4: Statistical comparison of genotype and alleles distribution of HSP70-1 (-110 A/C), (+190 G/C), HSP70-2 +1267 A/G, and HSP70-hom (+2437 T/C) polymorphisms among the three studied groups.|
Click here to view
|Table 5: Multivariate logistic regression analysis of risk factors for diabetic nephropathy adjusted for diabetes mellitus duration.|
Click here to view
HSP 70-1 +190 G/C polymorphism
The digestion products of Hsp70-1 gene + 190G/C polymorphism were 461 and 27 bp for GG and 488, 461, and 27 bp for GC and 488 bp for CC.
Regarding +190 G/C genotypes, there was a significant increased frequency of the CC genotype and C allele of +190 GC in Group I, GC in Group II, and GG in Group III compared to the other two groups. On comparing Group I with Group III, +190 GC polymorphism showed that CC genotype increased the risk of nephropathy by 6.7 folds and GC genotype increased the risk by 5.6 folds and C allele increased by 3.5 folds. On comparing Group I with Group II, the results showed that CC genotype increased the risk of nephropathy by 4.2 folds and GC genotype increased the risk by 1.5 folds and C allele increased by 1.9 folds [Table 4] and [Table 5].
HSP 70-hom (+2437 T/C)
The digestion products of HSP70-hom gene +2437 T/C polymorphism were 354; 273 bp for TT; 627, 354, and 273 bp for TC; and 627 bp for CC.
There were no significant differences in genotype and allele distributions of HSP70- hom (+2437 T/C) polymorphism among the studied groups.
HSP 70-2 +1267A/G
The digestion products of Hsp70-2 +1267 A/G gene polymorphism were 934 and 184 bp for GG genotype and 1118 bp, 184 bp, and 934 bp AG and 1118 bp for AA.
Regarding Hsp70-2 +1267 A/G gene polymorphism, there was highly significant increased frequency of the AG and GG genotypes and G allele in Group I and AA genotype in Group II. On comparing Group I with Group III Hsp70-2 +1267 A/G gene polymorphism, it was shown that G allele increased the risk of nephropathy by 5.60 folds. On comparison of Group I with Group II G allele, it was shown that there was an increased risk of nephropathy by 4.60 folds [Table 4] and [Table 5].
Haplotypes of the HSP70 genes were estimated. There was a statistically significant difference in the frequencies of four of these haplotypes, AGAT, AGGT, CGGT, and CCGT between Group I and Group III. While there was a statistically significant difference in the frequencies of AGGT and CGGT between Group I and Group II, no such significant differences were found between Group II and Group III except for CGAT haplotype [Table 5]. The results showed that, by multiple regression analysis after adjustment for DM duration, glycated Hb followed by gender, diastolic blood pressure, and age are independent risk factors for diabetic nephropathy (DN) [Table 5].
| Discussion|| |
DN is a major cause of ESRD., The aim of this study is to evaluate the association between HSP 70-1,2 and hom polymorphisms and susceptibility of Egyptian patients with Type 2 DM to develop DN. The present study results are in consistent with the result of Jeon et al who observed that there was no statistically significant difference as regards age and gender distribution among the three studied groups.
This study showed that there was a statistically significant difference between patients with Type 2 diabetes with and without nephro- pathy as regards the family history of diabetes and highly significant statistical difference when each of the patient groups (with and without nephropathy) was compared with the control group. This is as noticed by Bruce et al, Agarwal et al, and Themeli et al,,, who stated that in Type 2 diabetes, there is a well- documented association between a family history of the disease and its development.
In this study, there was a statistically significant difference among the patient groups and controls regarding BMI. This was explained by Qatanani and Lazar, who reported that the association between obesity and insulin resistance is likely a cause–effect relationship. On the other hand, it was reported that no clinically significant difference in the body weight and BMI was noted among diabetic patients and control groups.
The present study showed a highly statistically significant difference regarding blood level of urea and creatinine on comparing patients with nephropathy and those without. These results matched with the study of Wagle. On the other hand, Azar et al found no change in the kidney functions in normo- albuminuric patients with Type 2 diabetes.
The present study showed highly statistically significant difference regarding albumin/crea- tinine ratio in urine when comparing patients with nephropathy and those without. This was explained by 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 podo- cyte damage.
This study showed significant statistical difference regarding -110 A/C HsP 70-1 polymorphism with increased frequency of the CC genotype in patient group with nephropathy, increased frequency of AC genotype in patients without nephropathy, and increased AA genotype frequency in controls. The C allele of -110 A/C HSP 70-1 polymorphism is a risk factor for nephropathy in patients with Type 2 diabetes. Similar results were also noticed by Buraczynska et al who reported that highly significant differences in genotype distribution between diabetic patients with DN and healthy controls were observed. This study showed a significant statistical difference as regards +190 G/C genotype with increased frequency of the CC genotype in patient group with nephropathy, increased frequency of GC genotype in patients group without nephro- pathy, and increased GG genotype frequency in controls. The C allele of +190 G/C HSP 70-1 polymorphism is a risk of nephropathy. These results are in consistent with the results of Buraczynska et al who reported that significant differences in genotype distribution between T2DM patients with DN and healthy controls were observed.
Variations of -110A/C and +190G/C genotypes have been shown previously to be associated with Parkinson’s disease, celiac disease, and autoimmune thyroid disease.
This study showed a nonsignificant statistical difference regarding T/C HsP 70-hom polymorphism distribution on comparing the three studied groups with increased frequency of TT genotype in diabetic patients with DN and increased frequency of TC genotype in diabetic patients without DN. The studies by Buraczynska et al and Mir et al reported that there was no statistically significant difference in the genotype distribution between Type 2 DM patients with DN, those without DN, and healthy controls and also on comparing diabetic patients with DN and controls. On the other hand, these results were in contrast to the study of Umapathy et al who reported that evaluation of the Hsp70-hom polymorphisms by NcoI restriction digestion in Type 2 diabetes revealed that there was a significant increase of TT among patients and of CC among controls. In addition, Dhamodharan et al in their study showed that HSP70-hom T/T genotype is highly associated with patients with diabetic foot ulcer (DFU), and their functional polymorphism may play an important role in the pathogenesis of DFU in Type 2 diabetes in the South Indian population.
The current study showed that HSP70-2 gene variant at position +1267, a silent change in the coding region, was associated with nephro- pathy in Type 2 diabetic patients. This is consistent with the study of Buraczynska et al and Tahara et al, who found that the AA genotype of HSP70-2 had the highest level of mRNA expression among the three genotypes, whereas the GG genotype showed the lowest, suggesting that the A allele has a more protective potential than the G allele.
An association study based on haplotypes may have increased power for detecting disease associations; in the present study, there was a statistically significant difference in the frequencies of four of these haplotypes, AGAT, AGGT, CGGT, and CCGT between DM with DN and the healthy controls. There was a statistically significant difference in the frequencies of AGGT and CGGT between DM with DN and DM without DN. No such significant differences were found between DM without DN and the healthy controls except for CGAT haplotype; these results are comparable to the study of Zhang et al. Among the five common (>5% frequency) haplotypes (GAT, GAC, GGT, CAC, and CGT) assessed on coal workers with pneumoconiosis, two (GAT and GGT) showed significantly different frequencies between patients and the healthy controls.
This study concluded that the HSP 70-1 CC genotype of both -110 A/C and +190 G/C and the G allele distribution of HSP 70-2 +1267A/G is associated with the susceptibility to DN, while HSP70-hom +2437 T/C genotype distribution is not associated with the susceptibility to DN.
| Limitations|| |
The sample size is small because the kits for detection of gene polymorphism are very expensive to include more than that number of patients.
| References|| |
Luis-Rodríguez D, Martínez-Castelao A, Górriz JL, De-Álvaro F, Navarro-González JF. Pathophysiological role and therapeutic implications of inflammation in diabetic nephropathy. World J Diabetes 2012;3:7-18.
Girach A, Manner D, Porta M. Diabetic microvascular complications: Can patients at risk be identified? A review. Int J Clin Pract 2006;60:1471-83.
Buraczynska M, Swatowski A, Buraczynska K, Dragan M, Ksiazek A. Heat-shock protein gene polymorphisms and the risk of nephropathy in patients with Type 2 diabetes. Clin Sci (Lond) 2009;116:81-6.
Padmini E. Physiological adaptations of stressed fish to polluted environments: Role of heat shock proteins. Rev Environ Contam Toxicol 2010;206:1-27.
Kayani AC, Morton JP, McArdle A. The exercise-induced stress response in skeletal muscle: Failure during aging. Appl Physiol Nutr Metab 2008;33:1033-41.
Molvarec A, Tamási L, Losonczy G, Madách K, Prohászka Z, Rigó J Jr. Circulating heat shock protein 70 (HSPA1A) in normal and pathological pregnancies. Cell Stress Chaperones 2010;15:237-47.
Abid MR, Razzaque MS, Taguchi T. Oxidant stress in renal pathophysiology. Contrib Nephrol 2005;148:135-53.
Razzaque MS, Taguchi T. Involvement of stress proteins in renal diseases. Contrib Nephrol 2005;148:1-7.
Wu YR, Wang CK, Chen CM, et al. Analysis of heat-shock protein 70 gene polymorphisms and the risk of Parkinson’s disease. Hum Genet 2004;114:236-41.
Nam SY, Kim N, Kim JS. Heat shock protein gene 70-2 polymorphism is differentially associated with the clinical phenotypes of ulcerative colitis and Crohn’s disease. J Gastoenterol Hepatol 2007;22:1032-8.
Rifai N, Warnick R. Lipids, lipoproteins, apolipoproteins and other cardiovascular risk factors. In: Carl AB, Edward RA, David EB, editors. Tietz Textbook of Clinical Chemistry and Molecular Diagnosis. 4th ed., Ch. 26. Saunders; 2006. p. 918-22.
Shlipak M. Diabetic nephropathy: Preventing progression. BMJ Clin Evid 2010;2010:606.
Graham UM, Magee GM, Hunter SJ, Atkinson AB. Diabetic nephropathy and chronic kidney disease at a busy diabetes clinic: A study of outpatient care and suggestions for improved care pathways at a subspecialty specialist diabetic renal clinic. Ulster Med J 2010;79:57- 61.
Jeon YK, Kim MR, Huh JE, et al. Cystatin C as an early biomarker of nephropathy in patients with type 2 diabetes. J Korean Med Sci 2011;26:258-63.
Bruce DG, Van Minnen K, Davis WA, et al. Maternal family history of diabetes is associated with a reduced risk of cardiovascular disease in women with type 2 diabetes: The Fremantle Diabetes Study. Diabetes Care 2010;33:1477-83.
Agarwal N, Sengar NS, Jain PK, Khare R. Nephropathy in newly diagnosed type 2 diabetics 6): Lipids, lipoproteins, apolipo- proteins and other cardiovascular risk factors. In: Carl AB, Edward RA, David EB, editors. Tietz Textbook of Clinical Chemistry and Molecular Diagnosis. 4th ed., Ch. 26. Saunders; 2011. p. 918-22.
Themeli Y, Bajrami V, Barbullushi M, et al. Diabetic nephropathy and risk factors associated with DM in newly diagnosed type 2 diabetics. Endocrine Abstracts 2012;29:626.
Qatanani M, Lazar MA. Mechanisms of obesity-associated insulin resistance: Many choices on the menu. Genes Dev 2007;21: 1443-55.
Wagle TJ. Genderwise comparison of serum creatinine and blood sugar levels in type-2 diabetic patients. Bombay Hosp J 2010;52:20- 1.
Azar ST, Salti I, Zantout MS, Major S. Alterations in plasma transforming growth factor beta in normoalbuminuric type 1 and type 2 diabetic patients. J Clin Endocrinol Metab 2000;85:4680-2.
Brosius FC 3rd. New insights into the mechanisms of fibrosis and sclerosis in diabetic nephropathy. Rev Endocr Metab Disord 2008; 9:245-54.
Ramos-Arroyo MA, Feijoo E, Sanchez- Valverde F. Heat-shock protein 70-71 and HLA class II gene polymorphisms associated with celiac disease susceptibility in Navarra (Spain). Hum Immunol 2001 ;62,821-5.
Hunt PJ, Marshall SE, Weetman AP, et al. Histocompatibility leucocyte antigens and closely linked immunomodulatory genes in autoimmune thyroid disease. Clin Endocrinol (Oxf) 2001;55:491-9.
Mir KA, Pugazhendhi S, Paul MJ, Nair A, Ramakrishna BS. Heat-shock protein 70 gene polymorphism is associated with the severity of diabetic foot ulcer and the outcome of surgical treatment. Br J Surg 2009;96:1205-9.
Umapathy D, Krishnamoorthy E, Muthukumaran P, Rajaram R, Padmalayam I, Viswanathan V. Association of A1538G and C2437T single nucleotide polymorphisms in heat shock protein 70 genes with type 2 diabetes. LabMed 2012;43:250-5.
Dhamodharan UM, Ezhilarasi KM, Parthiban M, Rama R, Indira P, Vijay V. Is HSP70-hom (C2437T) Single Nucleotide Polymorphism (SNP) associated with Diabetic Foot Ulcer (DFU) among South Indian population? J Diabetic Foot Complications 2012;4:57-62.
Tahara T, Shibata T, Arisawa T, et al. The BB genotype of heat-shock protein (HSP) 70-2 gene is associated with gastric pre-malignant condition in H. pylori-infected older patients. Anticancer Res 2009;29:3453-8.
Zhang H, Jin T, Zhang G, Chen L, Zou W, Li QQ. Polymorphisms in heat-shock protein 70 genes are associated with coal workers’ pneumoconiosis in Southwestern China. In vivo
Osama Mohamady Elshahed
Department of Internal Medicine and Nephrology, Cairo University, Cairo
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]