Evaluation of miR-181a in Diabetes Mellitus Type-2 and its relation to Diabetic Nephropathy

microalbuminuria in DN group. Serum miR-181a significantly positively correlated with fasting insulin, fasting glucose, HOMA IR, ACR, serum IL-6 and BMI in DN group. The ROC curve analysis revealed high significant specificity and sensitivity of serum miR-181a, serum, and urine IL-6, and IL-18 suggesting their possible utility for the primary diagnosis of DN in DM. It is concluded that miR-181a may prove to be a valuable biomarker for DN prognostic and diagnostic applications.

Anti-inflammatory therapies have a great role nowadays in treatment of such chronic diseases, so studies to assess the levels of proinflammatory cytokines as interleukin IL-6 & interleukin IL-18 are essential [14].
IL-6 and IL-18 are associated with an increased inflammatory infiltrate with subsequent more severe kidney lesions [15].
Levels of IL-6 and IL-18 are strong predictors of diabetic nephropathy and their genetically down-regulated expression is associated with a favorable cardiometabolic profile [16].

Exclusion criteria:
The study excluded all patients with serum creatinine above 2.5 mg/dl, liver disorders, chronic inflammatory disease., infectious disease, patients with malignancy, type 1 diabetes, and other renal diseases.

At the clinic visit;
All patients were subjected to history taking.
Anthropometric measurements including waist and hip circumference in cm, weight in kg and height in cm were performed.The body mass index (BMI) has been calculated [34].

At the laboratory;
Estimation of estimate biochemical variables formula [35].

MiRNA Isolation
The miRNeasy Mini Kit (cat.no.217004, QIAGEN, Germany) was used to purify extracted total RNA, including miRNA, from the serum samples in accordance with the manufacturer's instructions.260 nm was the measurement point for the extracted RNA using spectrophotometry (JENWAY, USA).

Calculation of Relative Quantification (RQ) (relative expression)
Using the comparative cycle threshold (Ct) approach, the relative expression levels of mRNA and miRNA were determined in relation to the expressions of GAPDH and RNU6 snRNA.Using the 2-ΔΔCT method [36], the fold changes in mRNA and miRNA expression were determined.

demographic and clinical data
Table 1 showed highest levels of BMI, and disease duration in DN than other groups.

Levels of miR-181a in studied groups
When DN patients were compared to other groups, their levels of miR-181a, IL-6, and IL-18 increased significantly (Figure 1).Higher levels of miR-181a in DN patients with macroalbuminuria than DN patients with microalbuminuria (Figure 2).

Estimation of serum and urine IL-6, IL-18 levels in studied groups
Figure 3 showed that patients with DN showed significantly higher serum and urinary IL-6, IL-18 levels versus other groups.

Parameters in the subjects
Table 1 showed that there were significant

Correlations of miRNA 181a
In Table 2, miRNA 181a levels showed

Conclusion and Recommendations :
Diabetic nephropathy patients had higher serum levels of miRNA 181a.As opposed to non-diabetics, all diabetic patients had higher levels.Furthermore, patients with diabetic nephropathy who had macroalbuminuria had higher levels of miRNA 181a than those who had microalbuminuria.

Type 2
Diabetes Mellitus (T2DM) is a prevalent health problem that is most likely linked to obesity.People afflicted with T2DM are subject to complications involving micro and macro vasculature due to elevated glycemic level and insulin resistance [2].Diabetic Nephropathy (DN) occurs as a side effect to long standing hyperglycemia in T2DM individuals.DN occurs as a macrovascular complication due to macrovascular damage caused by glycation affecting renal vasculature.DN can eventually lead to end-stage renal disease due to poor glycemic control [1].Multiple factors contribute to the incidence of T2DM and its complications.Among these factors is insulin resistance which involves the inability of insulin to exert its actions throughout the body.Micro RNA (miRNA) is a type of RNA that post-transcriptionally regulates gene expression.Dysregulated miRNA has been suggested to contribute to conditions such as obesity and insulin resistance [3].miRNA 181a is one such miRNA that has been suggested to be linked to DM and more specifically to the incidence of DN in type 2 diabetic patients.miRNAs have the potential to become a new axis for understanding the pathogenesis and risk factors of diseases as well as contribute to new methods of management of such diseases [3].

(
fasting and post prandial glucose, HbA1c, insulin, c-peptide, total cholesterol, triglycerides, LDL-c, HDL-c, urea, creatinine) and 24 h urinary protein after an overnight fasting of at least 10 hours.For diagnosis of DM and diabetic nephropathy.Calculation of HOMA-IR on basis of fasting value of plasma glucose and insulin according HOMA model

(
p<0.001) increases in the mean levels of serum triglycerides, cholesterol, HBA1c, fasting glucose, fasting insulin, HOMA-IR, in diabetics with nephropathy and diabetics without nephropathy than those in the control group with the highest serum levels of triglycerides, cholesterol, HBA1c, fasting glucose, fasting insulin, HOMA-IR, levels in DN.It also showed that there were significant increases in the mean levels of Creatinine, urea, ACR in diabetics with nephropathy than those in other groups.
Fig 1. miRNA 181a levels in the studied groups.Significant (p-value <0.001) increases in miRNA 181a levels in patients with DN, and diabetic patients without nephropathy compared to control group, whereas they were higher in patients with DN compared to diabetic patients without nephropathy.

Fig 2 .
Fig 2. comparison between miRNA 181a levels between DN group's subgroups.Significant increases in miRNA 181a levels in patients with macro-albuminuria than those in patients with microalbuminuria within the patients with DN.

Fig 4 .
Fig 4. ROC Curve analysis for miRNA 181a, IL-6, and IL-18 in the early diagnosis of diabetic nephropathy in diabetic patients.

Furthermore,
In the current research, miRNA 181a mean levels exhibited a significant increase in diabetic patients withDN  and those without DN compared to those in control patients.The highest levels were observed in diabetic patients with DN.These results indicated a substantial relationship between miRNA 181a and the development and pathophysiology of diabetic nephropathy.This association was suggested by Zhang, Wu et al. (2018) shown to play an antifibrotic role through induction of fibroblast apoptosis and reduction of collagen type 1 expression.Further analysis revealed that miRNA 181-a targets the 3' untranslated region of fibroblast growth factor-1 in DN.Zhang, Wu et al. (2018) determined that overexpression of miRNA 181a could reduce synthesis of fibroblast growth factor 1 and its associated proteins thus contributing to renal fibrosis in DN.Liang and Xu (2020) determined that elevated miRNA 181a as a result of hyperglycemia is associated with the induction of cellular apoptosis.As a result, a new theory for DN in T2DM patients hinted that miRNA 181a might be implicated in Renal pathological changes.In addition, Liu, Chen et al. (2022) deduced from their research that, in the context of renal inflammation in DN, miRNA 181a demonstrates anti-inflammatory effects by targeting TNF-α.On the contrary, Zha, Qu et al. (2019) found that the influence of maternally expressed gene (MEG3) RNA through acting as a sponge for miRNA 181a led to downstream effects that promoted fibrosis and inflammatory response in DN.The current investigation also determined a strong positive correlation between miRNA 181a levels and fasting insulin, fasting glucose, & HOMA-IR levels in diabetics patients.The current outcome is further supported by Williams and Mitchell (2012) who found that elevated glycemic levels where associated with increased expression of miRNA 181a.Mononen, Lyytikäinen et al. (2019) as well determined that elevated https://ejmr.journals.ekb.eg/pathway leading to insulin resistance in T2DM through negatively correlating with genes directing the transfer of GLUT4 to the plasma membrane in the insulin signaling pathway which support the correlation reached in this research between elevated miRNA 181a levels and insulin laboratory results.Also, according to Zhou, Li et al. (2012) miR-181a could improve hepatic insulin sensitivity and glucose homeostasis, thereby offering a new therapeutic approach for treating insulin resistance and type 2 diabetes which further supports the theory that miRNA 181a plays a role in the pathogenesis of DM and its complications, most importantly DN.In the current investigation, it was discovered that patients with macroalbuminuria had mean levels miRNA 181a that were higher than those with microalbuminuria in diabetic patients with nephropathy.Additionally, in diabetic patients with nephropathy, there was a strong positive correlation between miRNA 181a and urine ACR.According to Gross, de Azevedo et al. (2005) who found that staging of diabetic nephropathy was dependent on the categorization of the degree of albuminuria exhibited by diabetic patients thus establishing a clear positive correlation in the present work between miRNA 181a levels and the degree of albuminuria in diabetic nephropathy revealed the association of miRNA 181a with the subsequent DN staging, and severity.Not only were miRNA 181a levels starkly distinguishable among diabetic patients with DN and DM patients without nephropathy but also distinguishable among macro and micro albuminuria patients with DN.The current study observed that the serum miRNA 181a showed high considerable sensitivity and specificity with extremely accurate diagnosis of diabetic nephropathy by using the ROC curve analysis for the early detection of diabetic nephropathy in diabetes mellitus.These findings which suggested utilization of serum miRNA 181a in the early diagnosis and progression monitoring of diabetic nephropathy in diabetes mellitus have not been previously studied.In the current study it was determined that diabetic patients generally exhibited higher BMIs than control patients.Furthermore, diabetic patients with diabetic nephropathy exhibited the highest BMIs which suggests that obesity can be associated with DN.This outcome was further supported by Tziomalos and Athyros (2015) being among the risk factors contributing to the incidence of DN.Kidneys affection in patients with diabetes mellitus is due to not only hyperglycemia, advanced glycosylation products but also the effect of proinflammatory cytokines.Both IL-6 and IL-18 are involved in multiple renal pathology e.g IgA nephropathy, lupus nephritis and diabetic nephropathy [17].Egyptian Journal of Medical Research (EJMR), Volume 5, Issue 1, January, 2024 127 https://ejmr.journals.ekb.eg/Diabetic nephropathy assessment is mandatory to give an effective reference for clinical immunotherapy researchers, who work to diagnose and improve the renal function in these chronic diseases [18].Assessment of the serum and urinary levels of IL-6, IL-18 in our study were done for the control group to know the normal values and for all diabetic subjects with and without nephropathy.Despite urine ACR measurement is the classical predictor for DN, pointing to the level of renal impairment and glomerular filtration membrane affection, urine protein can be affected by dietary habits and metabolic consumption of these patients.Therefore, urine ACR measurement is considered as a deficient tool for early recognition and prediction of DN.In contrast to IL-6 and IL-18 are significantly altered in the early stages of any kidney injury [19].Our results revealed a highly significant different in serum and urinary levels of IL-18, IL-6 comparative between both patients and control subjects.Meanwhile, serum and urinary IL-6 and IL-18 levels were higher in diabetic patients with nephropathy as compared with those without nephropathy.Wu R et al., 2018 research is in accordance with our results suggesting the involvement of IL-6 and IL-18 in the progression of diabetic complications.Also other researchers; Cui J et al., 2020, Ishikado et al., 2020 and Zhang et al., 2021 agreed with our results reporting that both IL-6 and IL-18 in serum and urine are associated with microalbuminuria and clinical albuminuria in diabetic patients.These researchers also stated that their levels are increased compared with diabetic patients who are without albuminuria.Therefore, serum and urinary levels of IL-18, and IL-6 may have great pathogenic roles in diabetic nephropathy and are good predictors for urinary albumin excretion in diabetes mellitus.Hadeel et al., 2021 stated that, besides the metabolic and hemodynamic factors, it is important to consider the great participation role of inflammatory cytokines on both pathogenesis and development of diabetic nephropathy.Hong., 2022 reported that there are major changes in the cellular proinflammatory cytokines e.g IL-18, and IL-6 in patients with diabetes as they play a significant critical role in the pathogenesis of diabetic complications through multiple biochemical and hemodynamic pathways.Emanuela et al., 2017 proved that the expression of IL-6 and IL-18 were increased more in patients with diabetic nephropathy and with overall disease exacerbation.This research explained that by the ability of both IL-6 and IL-18 to activate of numerous inflammatory signaling pathways; promoting the disruption of both the charge barrier and physiological barrier of the renal filtration https://ejmr.journals.ekb.eg/membrane allowing the leakage of urinary protein.Serum and urinary markers of IL-6 and IL-18 had a higher sensitivity and specificity compared to miRNA181a.LaPierre et al., 2017 stated AUC cutoff values of both miR-181a, miR-144 expression levels had a considerable specificity and sensitivity, as these diagnostic biomarkers for diabetes are varying according the glycemic control status of the patients.Meanwhile, a study done by Anker et al., 2012 concluded that there are a great antiinflammatory actions of miR-181a by targeting and blocking the TNF-α in the context of renal inflammation in DN.This were illustrated by Tang et al., 2020 who stated that the increased serum levels of IL-18 and IL-6 in diabetes mellitus didn't reflect only the chronic inflammation but it consider apart from atopic and autoimmune conditions.This was in concordance with the study of Tuttle et al., 2022 who stated that IL-6 and IL-18 are the most abundant cytokines associated with metabolic disorders.It causes major toxic effects on beta cells by receptormediated signaling inducing cell apoptosis.Regarding the Pearson Correlation, our results showed there is a important significant negative correlation between miRNA 181a and serum IL-6.This explained by Sheinerman et al., 2018 who stated that increased levels of miR-181a could be seen in both inflammatory conditions, beside the miR-181a has a role as an anti-inflammatory miRNA, likely depending on the cellular and physiological context of the patients.Moreover, Karlsson et al., 2020 reported that miR-181a play an important significant role in the aging process; identifying it as multimorbidity-associated miRNA.Therefore, it can be used as a biomarker of health status because it has different effects at different ages.