Alarming statistics shows a constant increase in the number of people with type 2 diabetes mellitus (T2DM) – by 49% over a 30-year period (Safiri et al., 2022) reaching a total of 529 million cases in 2021 (GBD 2021 Diabetes Collaborators, 2023). An increase in the T2DM mortality rate and associated complications is even greater – the number of deaths increased by almost 150% over the same 30-year period (Zhu et al., 2022). Since diabetic nephropathy (DN) is one of the most common complications of diabetes and a devastating chronic progressive condition, T2DM prevalence and mortality are directly associated with a global increase in the number of DN-related deaths (Thomas, 2019).

In order to prevent and control T2DM and related DN, as well as to develop novel, advanced and targeted therapeutic strategies, research tasks are focused on the investigation of molecular mechanisms and etiological factors underlying these multifactorial diseases, including roles of nitric oxide (NO) and insulin signaling (Krol and Kepinska, 2021). Numerous research and review articles were published describing their networking, linking vascular and metabolic diseases such as hypertension, energy dysbalance, obesity, T2DM and related complications, such as dyslipidemia, atherosclerosis, stroke, cancer. It seems that the regulation of metabolism is highly dependent on the vascular function, while insulin binding to its receptor on endothelial cells stimulates signaling cascades that balance between synthesis/activation and inhibition of endothelial NO synthase (eNOS or NOS3), which is the principal regulator of NO production in the vasculature. Therefore, insulin signaling contributes to vasorelaxation or vasoconstriction by acting on the eNOS activity (Levine et al., 2012; Krol and Kepinska, 2021). On the other hand, NO stimulates insulin uptake and transport by vascular endothelial cells, while endothelial dysfunction and protein S-nytrosylation in endothelial cells contribute to insulin resistance (IR) (Wang et al., 2013).

The expression and the activity of eNOS and, thus, endothelial function and IR, seem to be associated with functional NOS3 gene polymorphisms. The commonly analyzed variants, such as missense variant rs1799983 and the intronic VNTR rs869109213, are functionally well characterized. Namely, rs2070744 plays a role in transcriptional regulation of NOS3 expression by altering the binding sites for transcriptional regulators, while rs1799983 introduces amino acid substitution that alters eNOS conformation and influences its stability, interactions and localization (Elakkad et al., 2017; Shaheen et al., 2021). VNTR variant rs869109213 also displays a negative effect on eNOS biogenesis by exerting a negative feedback regulatory loop based on the RNA interference mechanisms (Zhang et al., 2008). Naturally occurring genetic variants within NOS3 gene which encodes eNOS have been investigated in relation to a number of T2DM complications affecting different organs as a consequence of high glucose concentration and improper vascular function (Oliveira-Paula et al., 2017; Li et al., 2022). These studies assessed biomarker properties of NOS3 variants in T2DM and secondary complications (including DN) in an effort to identify potentially useful genetic indicators for an early diagnosis, the assessment of genetic predisposition and prevention, as well as for the prediction of clinical progression of diabetes and microvascular complications. The findings, however, together with previous data syntheses (Dong et al., 2018; Varghese and Kumar, 2019; Sun et al., 2021; Li et al., 2022) were inconsistent or even conflicting, leaving the potential link between common NOS3 polymorphisms and T2DM-DN susceptibility highly inconclusive. Some of the potential reasons for the observed discordances include the small number of studies from certain ethnic groups in previous pooled analyses and a strong dysbalance in ethnic composition of data entries, small sample size of early original studies on the topic of interest, extreme between-study heterogeneity, differences in criteria for inclusion/exclusion of studies and variable diagnostic criteria for diabetic nephropathy. Therefore, the updated overview on the effect of NOS3 gene polymorphisms would be beneficial, provided that the influences of potential confounders are evaluated and that additional original studies from various ethnicities are included, following a strict predefined protocol with well elaborated and scientifically sound inclusion/exclusion criteria.

In an effort to overcome limitations disabling firm conclusions, mainly due to the small sample size associated with poor study strength, we conducted a systematic data synthesis on the effects of NOS3 polymorphisms on DN susceptibility in patients affected by T2DM. This updated meta-analysis incorporates novel findings from eligible studies assuring high accuracy of the pooled results and a comprehensive assessment of the potential effects of the suspected major confounding factors, such as ethnicity and criteria for patient selection, on the results of genetic association tests. Furthermore, the analysis for various genetic models of association was conducted in order to maximize the power of the study to detect significant associations.