The role of genetic factors in the development of recurrent urolithiasis

INTRODUCTION

The number of cases of urolithiasis in the world has increased by 48.57% over the past three decades, and worldwide mortality due to it has increased by 17.12% [1]. The prevalence of this disease on the territory of the Russian Federation over 15 years (2005–2019) in all the regions has become 16.20% higher [2].

Urolithiasis is a polyetiological disease of the urinary system, when salts are accumulated in the urinary tract [3, 4]. The pathogenesis of the disease is also based on genetic factors [5]. While studying the hereditary factor by the twin method, it was revealed that in patients with risk factors for urolithiasis, the heritability of the development of clinical disease forms was 45.00 – 50.00% [6]. In particular, single nucleotide polymorphism (SNP) in various genes may affect the risk of development and recurrence of this disease [7]. Early detection of genetic predisposition to this disease manifestation and recurrence may be the most important link in urolithiasis prevention [8]. Thus, the study of the role of polymorphisms in urolithiasis pathogenesis is an urgent task.

In this work, SNPs in TNFRS11B (Tumor necrosis factor receptor superfamily member 11B, or osteoprotegerin; rs3134057), ESR1 (Estrogen receptor isoform 1; rs851982), VDR (Vitamin D receptor; rs1540339), CASR (Calcium-sensing receptor; rs2202127), KL (Klotho protein; rs526906), whose protein products are somehow involved in maintaining calcium homeostasis or in the development of nephrological pathologies, were selected. Thus, TNFR-mediated (Tumor necrosis factor receptor) signaling is necessary for the adhesion of calcium oxalate crystals to the luminal membrane of the renal tubules, which is the main initiating mechanism of oxalate nephropathy [9], and the therapeutic TNFR blockade inhibits the development of progressive forms of nephrocalcinosis in this disease [10]. In postmenopausal women taking estrogen hormone replacement therapy, urine pH increases and calcium phosphate content is reduced compared to those who do not take estrogen drugs after menopause [11], which confirms the role of estrogen receptors in calcium homeostasis. The protein product of the VDR gene naturally regulates the biological activity of vitamin D, an important regulator of the calcium balance in the body. Thus, patients receiving excessive doses of vitamin D are more likely to suffer from urolithiasis [12]. The physiological effects caused by CASR are extensive, among them slowing the reabsorption of calcium ions in the nephron, increasing the excretion of water, citrate, protons, and reabsorption of phosphate ions. According to the literature, the presence of polymorphisms that reduce the CASR density on cells is associated with more frequent development of nephrolithiasis [13]. The KL forms a negative feedback loop with the mechanism of calcitriol synthesis, the most biologically active form of vitamin D. Mice deficient in this protein were characterized by an increased concentration of calcium in blood plasma [12]. In addition, KL can directly interact with non-selective TRPV5 (Transient receptor potential cation channel subfamily V member 5) cation channels involved in transcellular calcium reabsorption by distal nephron tubules [12].

The study aimed to explore the SNP associations (rs3134057, rs851982, rs1540339, rs2202127, rs526906) with the development of recurrent urolithiasis.

MATERIALS AND METHODS

Study design. The study was approved by the Ethics Committee of the Medical Scientific and Educational Center, Lomonosov Moscow State University (Protocol No. 12/20 dated December 21, 2020) and conducted in accordance with the postulates of the Declaration of Helsinki. All participating patients provided signed forms of voluntary informed consent. In total, 96 people were included in the study between January 2021 and January 2022; among them, 45 patients suffered from urolithiasis recurrence, and from the localization of the main concretion in one of the ureters (the main group); the control group consisted of 51 volunteers who did not suffer from urolithiasis, whose family history was also not burdened by this disease. The clinical and demographic characteristics of the study participants are presented in Table 1. The stone size was assessed using computed tomography of the urinary system organs without contrast enhancement.

Biomaterial sampling and studying. Venous blood samples were taken in a volume of 4.5 ml in vacuum tubes («Greiner Bio-One GmbH», Kremsmünster, Austria, 2016). Intact blood was aliquoted in 1.5 ml tubes and stored at -20 °C. After defrosting, each blood sample was thoroughly mixed by pulse vortexing. DNA was isolated from 200 µl of intact blood using QIAamp DNA Blood Mini Kit reagents («Qiagen GmbH», Hilden, Germany, 2006) using the QIAcube robotic system («Qiagen GmbH», Hilden, Germany, 2016) according to the manufacturer's recommendations.

Genotyping. The analysis of SNPs in the studied genes of patients of the main and control groups was carried out by means of using real-time PCR on a Real-time CFX96 Touch amplifier («Bio-Rad Laboratories, Inc.», Hercules, CA, USA, 2018) with the following thermal cycling conditions: exposure at 95 °C for 10 minutes, 40 cycles of denaturation at 92 °C for 15 seconds, and annealing/elongation at 60 °C for 1 minute. Information on the SNPs studied and the reagents used for their detection is presented in Table 2.

Statistical analysis. The analysis was carried out using the software SNPStats («Institut Català d'Oncologia», Barcelona, Spain, 2006) and IBM SPSS Statistics 22.0 («IBM Corp.», New York, NY, USA, 2013). Binomial logistic regression served as a statistical model for data analysis. A statistically significant difference was p < 0.05.

RESULTS

As a result of statistical analysis, an association was found between the presence of SNP rs3134057 in the TNFRS11B gene (odds ratio (OR) – 1.92; confidence interval (CI): 1.05–3.52; p = 0.031) and the development of recurrent urolithiasis, while no such statistically significant association was found for the other studied SNPs in the ESR1, CASR, VDR, and KL genes (rs851982, rs2202127, rs1540339, rs526906, respectively) (p > 0.05) (Table 3, Fig.).

To find an association of combinations of the SNPs mentioned above with the development of recurrent urolithiasis, an analysis of the non-equilibrium coupling of alleles was performed, which showed the absence of a statistically significant interaction between the SNPs studied (p > 0.05). A sub-analysis of haplotype combinations for 2 – 5 SNPs also did not reveal statistically significant combinations (p > 0.05).

DISCUSSION

All the SNPs studied are intronic. According to the Haploview service [15], the studied polymorphisms are located in regions with pronounced nonequilibrium coupling of alleles of their corresponding genes and can be associated with the degree of expression of the latter.

For some of the studied SNPs, their direct effects on metabolism and physiological processes in cell lines and multicellular organisms are known. Therefore, the introduction of the rs851982 ESR1 minor allele leads to an increase in the activity of the promoter of the ESR1 and RMND1 genes (required for the Meiotic Nuclear Division protein 1) in the cell lines ER+ MCF-7, BT-474 and ER+MCF-7, BT-474, ER−Bre-80, respectively (Estrogen receptor “+” Michigan Cancer Foundation-7; Breast tumor 474; Estrogen receptor “–“ breast-80); chromatin immunoprecipitation showed that the gene with rs8519882 SNP was enriched with GATA3 (GATA binding protein 3), but the GATA3 planting density did not differ between cells with SNP and normal allele [16]. In addition, the presence of rs851982 SNP is statistically significantly associated with a higher mineral density of the femoral neck (p = 0.012) [17]. Despite these data, the authors found no connection between rs851982 and recurrent urolithiasis development, which is consistent with the data of domestic scientists [18].

The presence of the minor allele rs1540339 of the VDR gene (both in the heterozygous variant and in the homozygous one: OR = 0.55; 95% CI: 0.35 – 0.88; p = 0.01 and OR = 0.404; 95% CI: 0.20 – 0.78; p = 0.005, respectively) reduces susceptibility to tuberculosis. In addition, tuberculosis patients have a significantly smaller amount of VDR mRNA. Both facts may indicate the influence of rs1540339 on the expression of the VDR gene. It is important to note that VDR plays a key role in the development of the immune response to infection, triggering the assembly of the phagolysosomal complex after binding to the active form of vitamin D [19]. However, data on the role of rs1540339 in the development of urolithiasis are contradictory, as there are works both confirming [20] and refuting its role [21]. In this study, no such connection was also found, and perhaps a further study of the effect of rs1540339 on the patient's predisposition to urolithiasis is required.

Up to date, there are no data from in vivo and in vitro studies for the rs2202127 SNP in the CASR gene demonstrating a change in expression in the presence of a minor allele. At the same time, it is known that the presence of rs2202127 is associated with an increased level of serum calcium [22], which may indirectly confirm its role in calcium metabolism.

Apolikhin et al. (2016) found an association of recurrent urolithiasis with rs2202127 SNP polymorphism in the CASR gene [18]. However, in the course of subsequent studies and in the course of the present study, these data were not confirmed [20]. Therefore, the existing data on the effect of rs2202127 SNP on recurrent urolithiasis are contradictory, indicating the need for larger studies with the allocation of additional subgroups of patients.

In this and similar studies, it was not possible to detect the association of rs526906 SNP in the KL gene with the development of urolithiasis [19][22], although it was shown that the presence of polymorphism in the KL gene led to an increase in the expression of the latter [23].

Currently, there are no data from in vivo and in vitro studies on changes in the expression of the TNFRS11B gene in the presence of rs526906 SNP; however, this polymorphism was associated with a reduced mass of the distal part of the radius [24]. The authors of this paper found that the presence of TNFRS11B polymorphism was associated with the development of urolithiasis, while in the study by Apolikhin et al. (2016), the indicated association was not found [18], which may be explained by differences in the inclusion conditions: only patients with recurrent urolithiasis, as well as the localization of the ureteral stone, were included in the present study.

In addition to the SNPs investigated in this article, there are other promising polymorphisms for studying genetic predictors of the development of urolithiasis. For example, there is evidence of a statistically significant association of polymorphism of the ORAI1 (Calcium release-activated calcium modulator 1) gene (rs7135617) with the risk of calcium oxalate stones [20].  ORAI1 is a subunit of the calcium membrane channel, which is activated when calcium reserves are depleted. Chou et al. (2011) identified two SNPs of this gene, rs12313273 and rs6486795, which increase the risk of developing calcium nephrolithiasis [25].

Limitations. A limitation of this study is the small sample size of patients.

CONCLUSION

The study of genetic predictors of urolithiasis development is an important and promising scientific direction in the context of trends toward a more personalized approach in modern medicine. Knowledge of the genetic predisposition to the disease in a particular patient can allow timely preventive measures and attention to modifiable risk factors, which can later significantly improve the patient’s quality of life, as well as improve economic indicators in the health sector.

In connection with the discovery of an association between the presence of rs3134057 SNP in the TNFRS11B gene and the development of recurrent urolithiasis, an urgent task is to study the effect of this SNP on the serum level of osteoprotegerin, a product of the TNFRS11B gene.

Key moments:

1. Osteoprotegerin plays an important role in the pathogenesis of recurrent urolithiasis; therefore, monitoring its level in various biomaterials may be of practical interest.
2. The presence of the minor allele rs3134057 SNP in the TNFRSF11B gene is significantly associated with recurrent urolithiasis development; genotyping for this polymorphism may be applied as part of the assessment of genetic predisposition to this disease.
3. The location of rs3134057 SNP in the intron of the TNFRS11B gene indicates its effect on the direct expression of osteoprotegerin, and not its structure. Thus, in future studies to study the level of osteoprotegerin in recurrent urolithiasis, to increase diagnostic accuracy, it is recommended to establish individual threshold values for each of the genotypes according to rs3134057