Single Nucleotide Polymorphisms of the RAC1 Gene as Novel Susceptibility Markers for Neuropathy and Microvascular Complications in Type 2 Diabetes

Single nucleotide polymorphisms (SNP) in the RAC1 (Rac family small GTPase 1) gene have recently been linked to type 2 diabetes (T2D) and hyperglycemia due to their contribution to impaired redox homeostasis. The present study was designed to determine whether the common SNPs of the RAC1 gene are associated with diabetic complications such as neuropathy (DN), retinopathy (DR), nephropathy, angiopathy of the lower extremities (DA), and diabetic foot syndrome. A total of 1470 DNA samples from T2D patients were genotyped for six common SNPs by the MassArray Analyzer-4 system. The genotype rs7784465-T/C of RAC1 was associated with an increased risk of DR (p = 0.016) and DA (p = 0.03) in males, as well as with DR in females (p = 0.01). Furthermore, the SNP rs836478 showed an association with DR (p = 0.005) and DN (p = 0.025) in males, whereas the SNP rs10238136 was associated with DA in females (p = 0.002). In total, three RAC1 haplotypes showed significant associations (FDR < 0.05) with T2D complications in a sex-specific manner. The study’s findings demonstrate, for the first time, that the RAC1 gene’s polymorphisms represent novel and sex-specific markers of neuropathy and microvascular complications in type 2 diabetes, and that the gene could be a new target for the pharmacological inhibition of oxidative stress as a means of preventing diabetic complications.


Introduction
Over 500 million people worldwide are affected by type 2 diabetes (T2D), which, along with obesity, is the second most frequent endocrinological disease [1]. About 7.8 million people in the Russian Federation have diabetes, with T2D accounting for the majority of cases [1]. Type 2 diabetes is associated with a multitude of disorders in lipid, protein, nucleotide metabolism, and redox homeostasis, leading to long-term complications that made T2D the ninth cause of mortality in 2020 [2]. In particular, impaired redox homeostasis is thought to be an important pathological condition underlying oxidative stress that contributes to the initiation and progression of type 2 diabetes [3][4][5]. Hyperglycemia, an increase in reactive oxygen species (ROS) production with cytosolic NADPH oxidase, and a deficiency in key antioxidants such as reduced glutathione (GSH) are considered to be the major damaging factors that are responsible for the structural and functional alterations

Demographic, Clinical and Laboratory Characteristics of Patients
The demographic, clinical, and laboratory characteristics of the study patients are shown in Table 1. The majority of the T2D patients had diabetic neuropathy (92.3%) and diabetic retinopathy (71.2%). Other T2D complications included diabetic angiopathy of the lower extremities (65.9%), diabetic nephropathy (38.4%), diabetic foot syndrome (7.6%), and coronary artery disease (32.5%). The patients with the above complications had a significantly longer duration of T2D (p = 0.001). As can be seen from Table 1, there were no regularities or trends in the quantitative parameters of redox homeostasis, such as glutathione or hydrogen peroxide, regardless of the type of diabetes complication.

Association of RAC1 Gene Polymorphisms with Diabetic Retinopathy
The frequency of the minor allele rs7784465-C was significantly higher in the patients with DR within the entire group (OR 1.35, 95%CI 1.09-1.68, p = 0.006) and in males (OR 1.52, 95%CI 1.05-2.21, p = 0.032) after a sex-stratified analysis. The alternative allele rs836478-T was associated with DR in the entire group of patients with DR (OR 1.34, 95%CI 1.14-1.59, p = 0.0005), in males (OR 1.51, 95%CI 1.15-1.99, p = 0.003), and in females (OR 1.27, 95%CI 1.02-1.57, p = 0.03). The genotype frequencies of the studied SNPs in diabetics with and without DR are shown in Table 2.  The genotypes rs7784465-T/C and rs836478-T/T were associated with the risk of diabetic retinopathy in the entire group of diabetics. A sex-stratified association analysis showed that the polymorphisms rs7784465 and rs836478 were associated with an increased risk for DR in males, whereas in females, no difference in the genotype frequencies for these SNPs was seen between the patients with and without DR. The estimated frequencies of the RAC1 haplotypes in T2D patients with and without DR are shown in Table 3.  The frequency of the haplotypes H2 rs4724800A-rs7784465C-rs10951982G-rs10238136A-rs836478T-rs9374G and H3 rs4724800G-rs7784465T-rs10951982A-rs10238136A-rs836478T-rs9374A was significantly higher in the patients with DR. A sex-stratified analysis showed a much stronger association of the haplotype H2 rs4724800A-rs7784465C-rs10951982G-rs10238136A-rs836478T-rs9374G with DR in diabetic males (OR 2.32, 95CI 1.46-3.67, p = 0.0004).

Polymorphisms of the RAC1 Gene and Diabetic Nephropathy
The minor allele rs836478-T was associated with DNF exclusively in males (OR 1.43, 95% CI 1.05-1.96, p = 0.025). The genotype frequencies for the studied SNPs in the diabetics with and without DNF are shown in Table 4.  As can be seen from Table 4, the polymorphism rs836478 was associated with the risk of DNF in males in the codominant model. However, the rs836478-C/T-T/T genotypes of RAC1 were found to be associated with an increased risk of diabetic nephropathy in male diabetics (OR 1.84, 95% CI 1.06-3.19, p = 0.024) in the dominant model. The other SNPs of the RAC1 gene showed no significant associations with a DNF risk. A haplotype analysis (Supplementary Table S1) revealed that none of the RAC1 haplotypes were associated with diabetic nephropathy.

RAC1 Gene Polymorphisms and the Risk of Diabetic Neuropathy
The frequencies of the minor alleles rs7784465-C (OR 1.80, 95% CI 1.17-2.75, p = 0.007) and rs836478-T (OR 1.35, 95% CI 1.02-1.80, p = 0.037) were significantly higher in the patients with DN compared to the patients without DN. The allele rs7784465-C was also associated with DN in females (OR 2.02, 95% CI 1.08-3.76, p = 0.028). The genotype frequencies of the RAC1 gene polymorphisms among the T2D patients with and without diabetic neuropathy are given in Table 5. The genotype rs7784465-T/C of RAC1 was associated with an increased risk of DN in the entire group of T2D patients and diabetic females. As can be seen from Table 6, the haplotype H2 rs4724800A-rs7784465C-rs10951982G-rs10238136A-rs836478T-rs9374G and the minor alleles rs7784465-C and rs836478-T were associated with an increased risk of DN in both diabetic males and females. Interestingly, the haplotype H5 rs4724800G-rs7784465T-rs10951982G-rs10238136A-rs836478C-rs9374G showed an association with an increased risk of DN only in males. Meanwhile, the haplotype H7 rs4724800A-rs7784465T-rs10951982G-rs10238136T-rs836478T-rs9374G possessed a protective effect against the DN risk in diabetic females. Entire group Global haplotype association p-value: 0.036

The Link between RAC1 Gene Polymorphisms to Diabetic Angiopathy of the Lower Extremities and Diabetic Foot Syndrome
The minor allele rs10238136-T was found to be associated with diabetic angiopathy of the lower extremities in females (OR 3.47, 95%CI 1.42-8.46, p = 0.004). The genotype frequencies for the RAC1 gene polymorphisms among the T2D patients with and without diabetic angiopathy of the lower extremities are given in Table 7. The genotype rs10238136-A/T was associated with an increased risk of DA in the entire group and in diabetic females, whereas the genotype rs7784465-T/C was associated with DA only in males. Meanwhile, the haplotype H7 rs4724800A-rs7784465T-rs10951982G-rs10238136T-rs836478T-rs9374G (Table 8) showed an association with an increased risk of DA in females. allele rs10238136-T (OR 3.67, 95%CI 1.48-9.10, p = 0.016) and haplotype H6 rs4724800A-rs7784465T-rs10951982G-rs10238136T-rs836478T-rs9374G (Table 9) were associated with diabetic foot syndrome in males.

Summary of the Study Findings
The present study found, for the first time, that the polymorphisms of the gene encoding Rac family small GTPase 1 (RAC1) in type 2 diabetes are associated with complications such as diabetic retinopathy, neuropathy, and angiopathy of the lower extremities. However, the observed associations were sex-specific. In particular, the genotype rs7784465-T/C was associated with an increased risk of retinopathy and angiopathy of the lower extremities in males, as well as diabetic neuropathy in females. Furthermore, the polymorphism rs836478 of RAC1 was linked to diabetic retinopathy and nephropathy in males, whereas the polymorphism rs10238136 was linked to diabetic angiopathy in females. Figure 1 depicts the structure of the RAC1 gene, the genomic position of the SNPs, and the haplotype structure of the gene, as well as summarizing the overall research findings. The RAC1 haplotypes were found to be associated with DR in males and with DN in females. Furthermore, the RAC1 haplotype rs4724800A-rs7784465C-rs10951982G-rs10238136A-rs836478T-rs9374G showed an association with DR in males and DN regardless of sex. In addition, the haplotype rs4724800A-rs7784465T-rs10951982G-rs10238136T-rs836478T-rs9374G was associated with a 4-fold risk of DA in females and DFS in males. The haplotype rs4724800A-rs7784465T-rs10951982G-rs10238136A-rs836478T-rs9374G showed an association with the increased plasma levels of ROS in diabetics with DR, DNF, and DN. The patients with DNF who carried the above haplotype had lower concentrations of total plasma glutathione. Moreover, the haplotype rs4724800A-rs7784465C-rs10951982G-rs10238136A-rs836478C-rs9374G was associated with higher ROS levels in patients with DNF, DN, and DA. The haplotype rs4724800G-rs7784465C-rs10951982A-rs10238136A-rs836478T-rs9374A was correlated with the increased ROS in patients with DFS, whereas the haplotypes rs4724800A-rs7784465C-rs10951982G-rs10238136A-rs836478T-rs9374G and rs4724800G-rs7784465T-rs10951982A-rs10238136A-rs836478T-rs9374A were correlated with the increased levels of total glutathione in the plasma. A functional annotation of the studied SNPs [20] showed that the minor alleles rs7784465-C, rs10951982-A, rs10238136-T, rs836478-T, and rs9374-A were associated with the increased expression of the RAC1 gene in various tissues and might be binding sites for transcription factors (TF). For instance, an analysis of the TF-binding affinity of the rs836478 polymorphism (which was associated with DNF) with the atSNP tool [26] (http://atsnp.biostat.wisc.edu/search, accessed on 2 November 2020) has shown that the minor allele rs836478-T was predicted to create binding sites for 34 TFs, including FOXC1, FOXD1, PBX1, GATA3, and POU3F3, which are enriched with GO terms that are related to the development of the nephron epithelium and renal tubules, as assessed by the STRING database [27] (https://string-db.org/, accessed on 14 December 2022).
Although many hypotheses have been proposed to explain the molecular pathways underlying diabetic complications, it is widely accepted that glutathione deficiency, the increased production of reactive oxygen species, and its resulting oxidative stress are the major pathological processes responsible for the development of diabetic complications [28][29][30][31][32].

Diabetic Retinopathy
Diabetic retinopathy is one of the most common complications of diabetes mellitus and is a major global contributor to vision loss and blindness [33,34]. According to a meta-analysis of large, population-based studies, the prevalence and progression of diabetic retinopathy have been linked to the serum levels of HbA1c, total cholesterol, and blood pressure, but only in about 10% of patients with type 2 diabetes [35], suggesting that other factors exist that explain the development of diabetic retinopathy in the majority of diabetics. Numerous studies [18,29,31,36] have shown that oxidative stress plays a key role in the onset of diabetic retinopathy. RAC1 is required for NADPH oxidase 2, an enzyme that generates reactive oxygen species. The transcriptional activity of the RAC1 gene may be regulated through epigenetic mechanisms. In particular, Kowluru and co-workers observed that the histone mark H3K9me3 at the Rac1 promoter assists Although many hypotheses have been proposed to explain the molecular pathways underlying diabetic complications, it is widely accepted that glutathione deficiency, the increased production of reactive oxygen species, and its resulting oxidative stress are the major pathological processes responsible for the development of diabetic complications [28][29][30][31][32].

Diabetic Retinopathy
Diabetic retinopathy is one of the most common complications of diabetes mellitus and is a major global contributor to vision loss and blindness [33,34]. According to a metaanalysis of large, population-based studies, the prevalence and progression of diabetic retinopathy have been linked to the serum levels of HbA1c, total cholesterol, and blood pressure, but only in about 10% of patients with type 2 diabetes [35], suggesting that other factors exist that explain the development of diabetic retinopathy in the majority of diabetics. Numerous studies [18,29,31,36] have shown that oxidative stress plays a key role in the onset of diabetic retinopathy. RAC1 is required for NADPH oxidase 2, an enzyme that generates reactive oxygen species. The transcriptional activity of the RAC1 gene may be regulated through epigenetic mechanisms. In particular, Kowluru and co-workers observed that the histone mark H3K9me3 at the Rac1 promoter assists with active DNA methylationhydroxymethylation reactions, activating Rac1 gene transcription in diabetic mice [19]. Cells that were exposed to high glucose concentrations were found to exhibit increased signaling in the chain Rac1-Nox2-ROS, increased levels of Rac1 transcripts, and increased 5-hydroxymethylcytosine levels at the gene promoter [37]. ROS overproduction has been shown to speed up the loss of capillary cells and to cause retinal neurodegeneration through mitochondrial damage, whereas the inhibition of ROS production was found to inhibit caspase-3-mediated neuronal apoptosis and to prevent vision loss [38,39].

Diabetic Nephropathy
Diabetic nephropathy is a clinical syndrome that is characterized by persistent albuminuria and a progressive decline in renal function [40]. DNF is thought to be the most common cause of end-stage renal disease, affecting 20% to 50% of people with diabetes. The mechanisms of DNF are very complex, and despite decades of intensive research, the pathogenesis of this complication in type 2 diabetes is still not fully understood [41,42]. Numerous pathways, processes, molecules, and conditions, such as oxidative stress, the renin-angiotensin-aldosterone system, mitogen-activated protein kinases, the formation of advanced glycosylation end products (AGE), connective tissue growth factor, transforming growth factor beta-1 (TGF-β), and inflammatory cytokines, are known to contribute to the onset and progression of DNF [43][44][45]. The pathways and mediators that are involved in kidney damage in type 2 diabetes share a lot of overlaps. For instance, it has been discovered that oxidative stress damages the kidneys through the activation of the reninangiotensin-aldosterone system, whereas angiotensin-II itself is capable of causing renal injury through oxidative stress [42]. Another example is NADPH oxidase stimulating the production of TGF-β, which stimulates the production of ROS via NADPH oxidase activation [41]. The experimental observation that the inhibition of oxidative stress improved a renal feature associated with streptozotocin-induced DNF has highlighted the role of oxidative stress in the induction and progression of DNF [46,47]. Meanwhile, oxidative stress can damage cells indirectly by activating other pathological pathways which damage the renal cells through unknown mechanisms [48]. Metabolic and hemodynamic alterations in the kidneys are also linked to oxidative stress, and both have additive detrimental effects on the organ [49].
The direct and indirect mechanisms by which oxidative stress causes kidney damage in diabetes have been proposed. ROS were found to cause direct damage to podocytes, mesangial cells, and endothelial cells, leading to proteinuria and tubule-interstitial fibrosis [50]. The mechanism of this damage was argued to be that the glomerulus, the filtering unit of the kidney, is more sensitive to oxidative injury than the other parts of the nephron [51]. Hyperglycemia is known to induce ROS production and oxidative damage to DNA, lipids, and proteins [52]. Chronic hyperglycemia can cause oxidative stress by increasing angiotensin-II levels, activating protein kinases, and increasing TGF-β expression [53]. For instance, increased angiotensin-II levels induce ROS production in the kidneys through the activation of NADPH oxidase [54]. It is observed that the ROS-associated activation of TGF-β causes the excessive remodeling of the extracellular matrix in the mesangium and promotes fibrotic processes in the kidneys [55]. As mentioned above, the increased production of ROS via NADPH oxidase in diabetes is attributed to the activation of the NF-κB pathway, which also promotes the transcriptional activation of the genes encoding inflammatory cytokines, thereby contributing to kidney injury and leading to renal fibrosis and a decline in renal function [56][57][58]. The activation of the α and β isoforms of protein kinases C is also known to induce oxidative damage to the kidneys through the increased production of NADPH-dependent superoxide anion radicals [59]. There are many other redox-sensitive signal transduction pathways, such as c-Jun N-terminal kinase (JNK), p38 MAP kinase, and the transcription factor activator protein 1 (AP-1), determining a vicious cycle between inflammation and oxidative stress [60,61].
We have established, for the first time, an association between the minor allele rs836478-T and an increased risk of diabetic nephropathy. According to the GTEx portal (https://gtexportal.org, accessed on 24 February 2023), the RAC1 gene is expressed at a relatively high level in the kidneys, suggesting an important role of the Rac family small GTPase 1 in this organ. There have been no studies on humans or animals that have investigated the expression level of the RAC1 gene in diabetic nephropathy, but there are studies that have investigated other NOX enzymes. In particular, an increased NOX-4 expression in renal cells was discovered in streptozotocin-induced diabetic rats [62], and subsequent studies have argued that upregulated NOX-4 is the primary source of the increased ROS production in the kidneys that contributes to renal fibrosis and DNF [63]. Both the deletion and the inhibition of the NOX4 and NOX1 genes have been shown to be renoprotective [64]. Finally, Ying and co-workers have recently observed that the binding of RAC1 to the pyrin domain containing 3 (NLRP3) activates the NLRP3 inflammasome in the kidney and accelerates the pathological processes underlying diabetic nephropathy [65]. The above studies clearly demonstrate the importance of RAC1-mediated oxidative stress for the development of diabetic nephropathy.

Diabetic Angiopathy of Lower Extremities
Diabetic angiopathy of the lower extremities is a change in the structure of the vessels of the legs in patients with diabetes mellitus, in the form of a decrease in the elasticity of the vascular wall and its thickening, leading to the narrowing of the lumen or the complete obliteration of the arteries. Increased oxidative stress is implicated in the pathogenesis of the various vascular complications of diabetes, including in diabetic angiopathy of the lower extremities [66][67][68]. It is well-known that abnormal endothelial-dependent vasodilation in diabetic patients is at least partially attributed to the reactive oxygen species that are primarily generated by up-regulated NOXs and downregulated endothelial nitric oxide synthase [69,70]. The increase in ROS levels and the decrease in nitric oxide are known to cause irreversible damage to the vascular endothelial cells through apoptosis [68]. The increased expression of NOX subunits, such as p22phox, p47phox, and p67phox, and the associated increased production of vascular superoxide anion radicals have been identified in diabetic subjects [71].

Diabetic Neuropathy
Diabetic neuropathy is a unique neurodegenerative disorder of the peripheral nervous system that preferentially targets sensory axons, autonomic axons, and later, to a lesser extent, motor axons [72]. The peripheral neurons that supply the feet are the longest cells in the body and require a properly functioning vascular supply, mitochondria, and glucose and lipid metabolism [73]. The duration of the diabetes and the plasma levels of the HBA1c are considered to be major predictors of diabetic neuropathy [74]. We revealed an association of the genotype rs7784465-T/C of the RAC1 gene with an increased risk of diabetic neuropathy in females. Female sex was found to be a risk factor for painful diabetic neuropathy, which is consistent with our findings [75]. The overproduction of superoxide anions has even been implicated in diabetic microvascular complications [76]. ROS production inhibits the GAPDH enzyme (glyceraldehyde-3-phosphate dehydrogenase) activity, causing upstream glycolytic metabolites to be diverted into the molecular pathways of glucose overutilization [77]. It is known that ROS production overwhelms the endogenous antioxidant defense in diabetic peripheral neuropathy, impairing the neural blood flow, nerve conduction, neurotrophic support, and neuronal mitochondrial function [78,79]. Hyperglycemia-induced oxidative and/or nitrosative stress causes DNA damage and the subsequent hyperactivation of poly(ADP-ribose) polymerases (PARP), which are the enzymes involved in DNA repair, cellular proliferation, and programmed cell death [80]. Overactivated PARPs consume NAD+, slowing glycolysis and impairing ATP function, as well as inhibiting GAPDH. PARP activation also promotes the formation of excess amounts of the superoxide anions and peroxynitrites that are associated with endothelial dysfunction, decreased nerve blood flow, neuronal energy deficit, a loss of nerve fiber density, and nerve conduction slowing [81,82].

Diabetic Foot Syndrome
Diabetic foot syndrome is a long-term complication of type 2 diabetes that is caused by a combination of vascular and neurological deterioration [83]. Epidemiological studies have shown that neuropathy is responsible for about 50% of the cases of diabetic foot syndrome [84]. Our study revealed that the RAC1 haplotype rs4724800A-rs7784465T-rs10951982G-rs10238136T-rs836478T-rs9374G was associated with a four-fold risk of DFS in males. The study of Rossboth S. and co-workers found a positive association of DFS with the male sex [85]. The pathogenesis of DFS has been linked to a variety of conditions, including oxidative stress, the malfunction of polyol and inositol metabolism, increased Na/K-ATPase activity, endoneural microvascular deficits and ischemia, defective axonal transport, and the non-enzymatic glycosylation of proteins in peripheral neurons [86,87].
The study has some limitations. Because the sample size of the patients with a specific diabetic complication was relatively small, the statistical power of the association analysis that was performed in the subgroups was decreased. A limited number of patients undergoing biochemical investigations of their redox homeostasis did not allow for the obtainment of more reliable estimates of the effects of the studied SNPs on these parameters in subgroups with particular diabetic complications. This limitation made it difficult to interpret the revealed associations between the RAC1 haplotypes and the plasma levels of the ROS and total glutathione. Furthermore, there may be other unexplored confounding variables in the diabetics that contribute to the development of diabetic complications.

Conclusions
The present study demonstrated, for the first time, that the genetic variants in the RAC1 gene represent novel susceptibility markers for diabetic retinopathy, nephropathy, angiopathy of the lower extremities, and neuropathy, with the potential to influence the risk of diabetic complications through perturbations in redox homeostasis. The sexual dimorphism of the associations between the RAC1 gene polymorphisms and the risk of diabetic retinopathy, particularly in men, appears to be due to the male sex itself being a known risk factor for this complication [85,88]. The mechanisms underlying the sex-specific associations of these genetic polymorphisms with a susceptibility for common diseases are a hallmark of research and continue to pique the interest of scientists [89,90]. The associations of the RAC1 gene haplotypes with the elevated concentrations of reactive oxygen species in patients with diabetic retinopathy, nephropathy, neuropathy, angiopathy, and diabetic foot syndrome may be intermediate damaging factors underlying the development of microvascular and nerve tissue diabetic complications. Because this is the first study to look into the role of the RAC1 gene polymorphisms in diabetic complications, there are no comparable studies to compare our findings to. Further studies into other populations of the world are required to validate these associations between the polymorphisms of the RAC1 gene and diabetic complications. However, our findings can already be applied to the development of new pharmacological agents that inhibit the RAC1 expression in specific tissues and thus reduce the ROS production.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biomedicines11030981/s1, Table S1: Estimated common haplotype frequencies of RAC1 gene in T2D patients with and without DNF; Table S2: Genotype frequencies for the studied RAC1 gene polymorphisms among T2D patients with and without diabetic foot syndrome (DFS); Table S3: Associations of RAC1 haplotypes with plasma redox homeostasis parameters.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Regional Ethics Committee of Kursk State Medical University (protocol No.10, date: 12 December 2016).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data supporting reported results are available upon request.