The Association between Single Nucleotide Polymorphisms in SIRT1 and ANGPT2 Genes and End-Stage Kidney Disease

Introduction

CKD represents a global health challenge with increasing morbidity and mortality rates.¹ This complex disorder progresses via intricate pathophysiological mechanisms, including oxidative stress, inflammation, and vascular dysfunction, which are significantly influenced by genetic factors.^2,3 Recent advances in molecular biology have highlighted two significant genes - SIRT1⁴ and ANGPT2⁵ in the kidney function and disease progression context.

SIRT1 is a highly conserved NAD⁺-dependent class III histone deacetylase.⁶ Predominantly located in the nucleus, it facilitates nuclear-cytoplasmic shuttling by regulating nucleosome histone acetylation and the activity of various transcription factors.^7,8 SIRT1’s protective effects extend to reducing cellular senescence through p53 deacetylation, particularly following DNA damage and oxidative stress.^9,10

SIRT1 deficiency in diabetic kidney disease, particularly in db/db mice podocytes, increases p65 and STAT3 acetylation, intensifying inflammatory responses and worsening proteinuria and kidney injury, highlighting its critical role in mitigating diabetic kidney damage.¹¹ Notably, single-nucleotide polymorphisms (SNPs) in the SIRT1 gene can influence its function and potentially affect CKD development and progression.²

SNP rs2273773 is in the intronic region of SIRT1, located in the intronic region on chromosome 10 (10q21.3). It is linked to the regulation of oxidative stress, inflammation, and aging, key factors in CKD progression, and is associated with abnormal cholesterol metabolism,¹² coronary artery calcification, and protective effects against vascular calcification.¹³ This suggests SIRT1 expression’s potential regulatory impacts on CKD pathogenesis.¹

Complementing SIRT1’s role, the Angiopoietin/Tie2 system, particularly Angiopoietin-2 (ANGPT2), is crucial in vascular stability and endothelial function.¹⁴ While Angiopoietin-1 promotes vascular quiescence and structural integrity through the PI3K/Akt pathway,¹⁵ ANGPT2 can disrupt endothelial function and vessel permeability, potentially leading to fluid overload in patients with CKD.⁵

SNP rs2442598, located in the intronic region of the ANGPT2 gene on chromosome 8 (8p21.2), is associated with disease susceptibility in systemic sclerosis and adverse outcomes in diabetic nephropathy, potentially influencing ANGPT2 expression and affecting endothelial function and vascular integrity, both critical in CKD and ESKD progression.^16,17

The molecular interplay between SIRT1 and ANGPT2 presents promising therapeutic potential.^1-18

Current research suggests that modulating SIRT1 expression and activity through specific inhibitors and activators, such as Ex-527 and resveratrol, may offer new therapeutic strategies for CKD management.⁴ Understanding these genetic variations and their impact on CKD progression could lead to more personalized treatment approaches and improved patient outcomes.^19,20

This study investigated the association between SIRT1 and ANGPT2 SNPs and CKD, and between their genotypes and clinical and laboratory parameters.

Materials and Methods

This cross-sectional study was conducted on 192 subjects of both sexes, ≥ 18 years old with ESKD, who have been undergoing dialysis for 6 months to 5 years, with a treatment frequency of 4 hours per session, three times per week. Individuals were divided into two groups: 96 patients diagnosed with ESKD and 96 healthy controls. The participants were recruited from the nephrology outpatient clinic and dialysis unit of Menoufia University Hospital, Egypt, between July 2023 and July 2024.

The exclusion criteria include those diagnosed with stages I-IV CKD, individuals with chronic liver or chest disease, morbidly obese or obese patients, individuals with cancer or autoimmune diseases, and patients with ischemic heart disease.

The study was approved by the Ethics Committee of the Institutional Review Board (IRB approval number: BIO14-1/7/23). Written informed consent was obtained from all participants.

A comprehensive medical history was obtained from each participant, followed by thorough general and clinical examinations. Laboratory investigations were performed for all subjects, including renal, hepatic functions, lipid profile, mineral, and hormonal parameters. DNA extraction for genotyping analysis was performed to determine the genetic variations in SIRT1 (rs2273773) and ANGPT2 (rs2442598) polymorphisms. The primary outcomes were the association between SIRT1 (rs2273773) and ANGPT2 (rs2442598) gene polymorphisms and the risk of developing ESKD. Secondary outcomes include examining the relationship between these genetic variants and clinical and laboratory parameters. Genotyping analysis was performed to determine the genetic variations in SIRT1 (rs2273773) and ANGPT2 (rs2442598) gene polymorphisms using TaqMan® allelic discrimination assay kit from (Biosearch, USA) [Figure 1].

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Figure 1:

Amplification plots for SNP genotyping in real-time PCR. Yellow line = the fixed ΔRn (normalized fluorescence-above-baseline) threshold above background; the cycle at which each red curve first crosses this line is recorded as that wells cycle threshold (Ct). Red lines = the amplification (ΔRn vs. cycle) for each reaction.

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Results

Primary causes of ESKD included diabetic nephropathy (45%), hypertensive nephrosclerosis (30%), and glomerulonephritis (25%). Biopsies were performed in 60% of patients with ESKD. Both groups showed comparable demographic characteristics. Laboratory differences in ESKD patients and control groups have been shown in Table 1.

The rs2273773 TT genotype was present in 58.3% of patients and 84.4% of controls. The rs2273773 TC genotype was present in 31.3% of ESKD patients and 13.5% (13/96) of controls (OR= 3.338). The rs2273773 CC genotype, with highest risk association, was found in 10.4% of patients and 2.1% of controls (OR= 7.232). Hardy-Weinberg equilibrium was maintained in ESKD patients and controls, suggesting no deviation from expected genotype frequencies. Analysis of inheritance models showed a dominant effect for rs2273773 TC+CC vs. TT (OR=3.857, p < 0.001), and a recessive effect for rs2273773CC vs. TT+TC (OR=5.465, p = 0.031). Co-dominant models further confirmed the elevated risks for rs2273773TC vs. TT (OR: 3.338) and rs2273773 CC vs. TT (OR: 7.232). Allele frequency analysis demonstrated a higher T-allele prevalence in controls (91.1%) compared to patients with ESKD (74%), whereas the C allele was more frequent in patients with ESKD (26%) than controls (8.9%) (OR=3.625, p < 0.001). These findings highlight a strong genetic association of the rs2273773 polymorphism with ESKD susceptibility [Table 2].

The rs2442598 TT genotype was present in 18.8% of patients and 58.3% of controls. The rs2442598 TC genotype was found in 43.8% of patients and 33.3% of controls (OR=4.083), suggesting an increased risk of ESKD. The rs2442598 CC genotype, demonstrating the strongest risk association, was observed in 37.5% of patients and 8.3% of controls (OR=14.0). Patients with ESKD and controls were in Hardy-Weinberg equilibrium, indicating no significant deviation from expected genotype frequencies. Inheritance model analysis showed a dominant effect for the rs2442598TC+CC vs. rs2442598 TT comparison, with 81.3% of patients and 41.7% of controls (OR 6.067, p < 0.001). The recessive model analysis rs2442598 (CC vs. TT+TC) further reinforced this association (OR 6.6, p < 0.001). Co-dominant models confirmed these findings, showing an OR of 4.083 and 14.0 for rs2442598 TC vs. TT and rs2442598 CC vs. TT, respectively. Allele frequency analysis revealed a higher T allele prevalence in controls (75%) compared with patients (40.6%), while the C allele was more common in patients (59.4%) than in controls (25%) (OR=4.385, p<0.001). This further supported the association between rs2442598 polymorphism and ESKD susceptibility [Table 2].

The associations between rs2442598 genotypes and various clinical parameters identify diastolic blood pressure as the sole parameter with a significant genotype-related variation [Table 3]. Results highlighted the potential role of specific haplotypes in ESKD susceptibility. They revealed the presence of specific haplotypes significantly more in patients and others in controls [Table 4].

Univariate analysis revealed significant associations between ESKD and lipid profile components (triglycerides, total cholesterol, HDL, LDL), calcium metabolism markers (total and ionized calcium), vitamin D levels, and the genetic variants rs2442598 and rs2273773. However, in the multivariate analysis, only two factors emerged as independent risk factors: vitamin D levels (OR: 0.854, p = 0.008) and the rs2442598 TC+CC genotypes (OR: 7.818, p = 0.009) [Table 5].

Allelic discrimination plots provide a qualitative representation of genotype clustering for rs2273773 and rs2442598, respectively [Figure 2].

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Figure 2:

Allelic discrimination plots.

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Discussion

CKD and its progression to ESKD represent significant public health challenges characterized by complex interactions between genetic predisposition and metabolic disturbances. Recent evidence suggests that genetic variations in SIRT1 and ANGPT2 may influence disease susceptibility and progression through various pathophysiological mechanisms.^21,22 Our study investigated the potential associations between SIRT1 rs2273773 and ANGPT2 rs2442598 polymorphisms and ESKD risk, revealing significant genetic and metabolic correlations that may contribute to disease development and progression. The rs2442598 polymorphism demonstrated strong associations with ESKD risk, with TC and CC genotypes. The rs2273773 polymorphism revealed significant correlations with LDL/HDL ratios, ionized calcium levels, and parathyroid hormone concentrations. Multivariate analysis identified vitamin D levels and rs2442598 TC+CC genotypes as independent risk factors for ESKD.

Both SNPs are located in intronic regions, which are known to influence gene expression through regulatory mechanisms such as transcription factor binding sites and expression quantitative trait loci.^23,24

The ENCODE data, accessible via the UCSC Genome Browser, can provide detailed information on the effect of these SNPs on chromatin architecture and gene regulation. For instance, rs2273773, located in SIRT1, has been associated with oxidative stress and inflammation, suggesting it may influence the expression of genes involved in these pathways.^25,26 Similarly, rs2442598 in ANGPT2 may affect endothelial function and vascular integrity, potentially through interactions with other genes in the Angiopoietin/Tie2 pathway.²⁷

Furthermore, non-coding variants like these can disrupt transcription factor binding sites, leading to allele-specific gene expression changes.²⁸ The identification of footprint quantitative trait loci associated with these SNPs can enhance our understanding of their functional consequences.²⁹

The distribution of rs2273773 genotypes revealed a significant association with ESKD risk, with TC and CC genotypes showing increased odds ratios compared to the TT genotype. These findings align with Kovanen et al.,³⁰ who reported significant associations between rs2273773 and cardiovascular parameters, particularly diastolic blood pressure. Moreover, these findings add to the growing body of evidence regarding SIRT1’s role in renal disease, though it differs from Shimoyama et al.’s³¹ findings, where rs2273773 was primarily associated with metabolic parameters in a Japanese population. Maintaining Hardy-Weinberg equilibrium in both groups strengthens the validity of our genetic associations.

Our analysis of rs2442598 demonstrated strong associations with ESKD susceptibility, with TC and CC genotypes showing significantly higher risk. This finding is exciting compared to Lan et al.’s³² study, which found that the TT genotype of rs2442598 was protective against coronary artery disease, suggesting potential shared genetic mechanisms between cardiovascular and renal pathologies.

The haplotype analysis revealed a predominant TT haplotype in controls (70.3%) vs. a CC haplotype in patients (38.5%), with significant linkage disequilibrium between rs2273773 and rs2442598. These findings complement the individual SNP analyses and suggest potential synergistic effects of these variants on ESKD risk. The linkage patterns, though modest, indicate potential evolutionary conservation of these genetic regions.

In the final multivariate analysis, vitamin D levels and rs2442598 TC+CC genotypes emerged as independent risk factors for ESKD. This finding is particularly noteworthy when considered alongside Tsai et al.’s⁵ observations regarding the prognostic significance of fluid status and ANGPT2 levels in CKD progression, suggesting multiple pathways contributing to renal disease progression.

Our findings revealed significantly elevated blood pressure values in patients with ESKD compared to controls, with notably higher systolic and diastolic measurements. These observations align with the established understanding of cardiovascular complications in ESKD. Interestingly, our genetic analysis revealed that the rs2442598 CC genotype carriers exhibited lower diastolic blood pressure than TT carriers. This finding contrasts with Zhong et al.,³³ who found that rs2273773 CT + CC genotypes were associated with lower blood pressure in hypertensive patients, suggesting different genetic influences on blood pressure regulation in ESKD vs. primary hypertension.

Our study demonstrated significant lipid profile alterations in patients with ESKD, characterized by elevated triglycerides and total cholesterol, with reduced HDLc. These findings parallel those of Altaher et al.³⁴ in premature myocardial infarction patients, suggesting shared metabolic perturbations across cardiovascular and renal diseases. Notably, the rs2273773 genotype is significantly associated with the LDL/HDL ratio, with CC carriers exhibiting the highest values (9.48 ± 8.54 mg/dL), indicating a potential genetic influence on lipid metabolism in ESKD.

The observed disturbances in calcium homeostasis, characterized by reduced calcium levels and elevated parathyroid hormone (PTH), align with Chang et al.’s²² observations of progressive mineral metabolism disruption in CKD. The rs2273773 genotype significantly influenced these parameters, with CC carriers showing the highest PTH levels and lowest vitamin D levels, suggesting genetic modulation of mineral metabolism in ESKD.

While the wild-type genotypes (TT) were more frequent in controls, the mutant genotypes (TC/CC) showed higher ORs for ESKD, indicating recessive effects. The small sample size limited the power to detect differences in mutant genotypes. Future studies with larger cohorts are needed to confirm these associations.

This study has several limitations, including its cross-sectional design and a single center. The findings may not be generalizable to other populations. The study focused on two specific gene polymorphisms, which may not capture ESKD’s full genetic complexity. Potential confounding factors and interactions between genetic variants were not comprehensively explored. Future studies with larger cohorts are needed. Longitudinal studies tracking CKD progression to ESKD could provide valuable insights into the temporal relationship between these genetic variants and disease progression. Investigation of additional SNPs in the SIRT1, ANGPT2, and other candidate genes involved in renal pathophysiology would provide a more comprehensive understanding of ESKD’s genetic architecture. Functional studies examining the molecular mechanisms by which these SNPs influence gene expression and renal function are warranted. Finally, exploration of gene-environment interactions and potential therapeutic implications of these findings could lead to personalized approaches for ESKD prevention and management.

To summarize, we did a cross-sectional study focusing on evaluating the association of gene polymorphisms and the risk of developing ESKD and its clinical and laboratory parameters. We have found that ESKD and some of its systemic consequences are significantly associated with SIRT1 and ANGPT2 polymorphisms.

We found that ESKD and its related systemic complications were significantly associated with SNPs in the SIRT1 (rs2273773) and ANGPT2 (rs2442598) genes.

ESKD is significantly associated with SIRT1 and ANGPT2 polymorphisms. Specifically, rs2442598 TC+CC genotypes and reduced vitamin D levels emerged as independent risk factors. The findings highlight the complex interplay between genetic variations, mineral metabolism, and renal dysfunction, suggesting potential genetic markers for disease susceptibility and progression.