MicroRNA-451 as an Early Predictor of Chronic Kidney Disease in Diabetic Nephropathy

Background Diabetes mellitus is the leading cause of end-stage renal disease worldwide. Microalbuminuria is the cornerstone for the diagnosis of diabetic nephropathy. However, it is an inadequate marker for early diagnosis. MicroRNAs are not only new and promising markers for early diagnosis but also, but they may also play a role in the prevention of disease progression. Methods This study included ninety patients with type 2 DM in addition to 30 control subjects. MicroRNA-451 expression in blood and plasma using real-time PCR was evaluated in addition to the classic diabetic nephropathy markers (serum creatinine, urinary albumin, and eGFR). Results There was a significant difference between the studied groups versus control regarding serum creatinine, eGFR, urinary, and plasma microRNA-451 with p=0.0001. Patients with eGFR 60 ml/min/1.73 m2 showed a significantly higher plasma microRNA-451 (29.6 ± 1.6) and significantly lower urinary microRNA-451 (21 ± 0.9) in comparison to patients with eGFR >60 ml/min/1.73 m2 and p=0.0001. eGFR showed a positive correlation with urinary microRNA-451 and negative correlation with both plasma microRNA-451 and urinary albumin. Both plasma and urinary microRNA-451 are highly sensitive and specific markers for chronicity in diabetic nephropathy patients with sensitivity of 90.9% and 95.5% and specificity of 67.6% and 95.6%, respectively. Conclusion MicroRNA-451 is a promising early biomarker for chronic kidney disease in diabetic nephropathy with high sensitivity and specificity.


Introduction
ere is an increased prevalence of diabetes mellitus (DM) all over the world. e major complications related to DM are attributed to the microvascular and macrovascular affections [1]. Diabetic nephropathy is one of the most common and serious complications in diabetic patients, as well as being the leading cause of end-stage renal disease worldwide [2]. Microalbuminuria is the cornerstone for early diagnosis of diabetic nephropathy. However, many studies have denoted this marker as an inadequate marker for early diagnosis, though it is easy and accessible [3].
Molecular techniques with different accessible detection of biomarkers have been developed to use the new biomarkers as a target for early diagnosis of diabetic nephropathy [4].
MicroRNAs play an essential role in regulating cell proliferation, differentiation, apoptosis, neoplastic transformation, and apoptosis. e role of miRNAs in DN development is through the development of renal fibrosis [12]. e studies hypothesized that some miRNAs lead to renal fibrosis through their effects on the transforming growth factor -β1 (TGF-β1) signal pathway and their impact on epithelial-mesenchymal transformation [13,14]. e principal function of TGF-β1 is the regulation of proliferation in the extracellular matrix, fibroblast, and myofibroblasts [15]. In renal damage, the numbers of TGF-β1 receptors increase with the initiation of fibrosis [16]. About twenty-two types of miRNAs were studied in diabetic nephropathy; fifteen were upregulated (miRNA-181b, miRNA-21, miRNA-30, miRNA-194, miRNA-215, and others). At the same time, seven of them were downregulated in the diabetic nephropathy group compared to control groups (miRNA-26a, miRNA-424, miRNA-126, miRNA-574-3p, miR-155, miR-223, and miR-192) [17]. Urinary exosomal miR-451-5p was only studied in diabetic rats as an early and sensitive noninvasive indicator of renal disease [18]. e advantages of the use of miRNAs as a diagnostic biomarker are their stability in blood and urine samples and being noninvasive markers. erefore, the quantitative measurement of miRNA in plasma and urine samples can be used as a diagnostic marker for monitoring the development of diabetic complications leading to the modification of the treatment with increased quality of life and the survival rates of the patients [19].
To the best of our knowledge, this is the first study that evaluated urinary and blood levels of miRNA-451 as an early biomarker for the diagnosis of diabetic nephropathy and chronic kidney disease in diabetic patients.

Materials and Methods
A case-control study including 90 diabetic patients recruited from outpatients' clinics from Mansoura University Hospital from January 2017 till November 2018 was conducted, in addition to 30 healthy subjects with normal kidney functions, normal blood glucose levels, and normal renal ultrasonography.
Patients were classified according to the presence of albuminuria into four groups: (i) Group 1, including 30 patients with normoalbuminuria and urinary albumin level <30 mcg/mg creatinine (ii) Group 2, including 30 patients with microalbuminuria and urinary albumin level between 30 and 299 mcg/mg creatinine (iii) Group 3, including 30 patients with macroalbuminuria and urinary albumin level >300 mcg/ mg creatinine (iv) Group 4, including 30 healthy individuals of the same age and sex e criteria for inclusion of the participant were age above 18 years with known DM type 2 and who are receiving their regular and routine antidiabetic medications, either orally or as an insulin therapy from the outpatients' clinics of Mansoura university hospitals with normal blood pressure, systolic blood pressure ≤140 mmHg and diastolic blood pressure ≤90 mmHg. Patients with congenital kidney diseases, malignancy, chronic glomerulonephritis, endocrinal diseases other than DM, and eGFR <30 ml/min/1.74 m 2 and those with any abnormal renal ultrasonography finding were excluded.
e Medical Ethical Committee of Mansoura Faculty approved the study. A voluntary informed consent was taken from all patients. Confidentiality and privacy were considered as regard personal, clinical, and laboratory data.
Each subject had a complete medical history recording and comprehensive physical examination.
Patients were reclassified according to the GFR level to the following: (i) Group 1, including 68 patients with eGFR >60 ml/ min/1.73 m 2 (ii) Group 2, including 22 patients with eGFR <60 ml/ min/1.73 m 2

Urine Samples.
A urine sample was collected in a clean container from each subject and transported to the laboratory for complete urine analysis, including amount registration, glucose, ketone, creatinine, and albumin measurement. Quantitative measurement of urinary albumin and creatinine was carried out by using biosystem kits (Biosystems S.A., Barcelona); after that, an aliquot of urine was kept at −80°C for measurement of miRNA-451.

Urinary
Albumin. e albumin in urine is measured by turbidimetry method as albumin binds to antihuman albumin coating agglutination latex particles. e quantity of albumin was measured at 505 wave length with RA analyzer 50.

Blood Samples.
A ten-milliliter blood sample was obtained from each subject after eight hours of fasting and divided into three aliquots. One aliquot was used for complete blood counts by Sysmex system and HBA1c by Stanbio kit (Boerne, Texas, USA, Cat. No. 0350) by the use of quantitative colorimetric method using column chromatography for determination of glycohemoglobin in whole blood. From the other aliquot, serum was separated and subjected to laboratory measurement of serum creatinine, albumin, cholesterol, total triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood urea nitrogen (BUN), and fasting blood glucose level by autoanalyzer Dialab (A-2351 Wr. Neudorf, Austria). Another blood sample was obtained after two hours for postprandial blood glucose levels. e serum was separated from the third aliquot and kept frozen at −80°C for the molecular study of miRNA-451 by a real-time polymerase chain reaction.

eGFR Measurement.
MDRD equation was used to calculate the eGFR for each subject: International Journal of Nephrology

RNA Extraction and Quantification.
MicroRNA-451 extraction from the plasma samples was performed by Qiagen miRNA Easy kit and from the urine samples by Norgen Urine microRNA purification kit, following the manufacturers' instructions. MicroRNA-451 concentrations and purity were determined with Nano-Drop 2000 UV spectrophotometer ( ermo Scientific, USA) using the A260/280 nm ratio.

Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR).
e expression of mature miR-451 was determined by qRT-PCR, as described in detail previously using the TaqMan Human MicroRNA Assay kit (Life Technologiescatalogue number #001141) and the real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) [20]. e endogenous internal control was RNU6B (Life Technologiescatalog number #4440887). All assays were performed in triplicate. Real-time PCR results were recorded as threshold cycle numbers (Ct) and were normalized against an internal control (U6 RNA) and then expressed as fold changes. e miRNA expression was quantified as δCt values, where Ct � threshold cycle, δCt � (Ct target microRNA − Ct RNU6B).

Statistical Analysis
Statistical analysis was carried out by the use of SPSS 22. Numerical Data are presented as mean ± SD for normally distributed values. ANOVA analyzed differences between the groups. Pearson's correlation coefficient was employed to test the correlations between different variables. Receiver operating characteristics (ROC) analysis was used to calculate the area under the curve (AUC) for microRNA-451 levels of serum and urine to find the best cutoff values for identifying renal impairment in diabetic patients. All results were considered significant if p < 0.05.

Results
In the current study, patients were classified according to the level of albuminuria into three groups. ere was no significant difference between the studied groups as regard age, sex, WBCs, platelets, and HDL (Table 1).
On the other hand, there was a significant difference in the levels of fasting blood glucose, creatinine, BUN, HBA1c, and hemoglobin with p � 0.0001. Total cholesterol, LDL, and TGs showed a significant difference between the studied group with p-value, 0.0001, 0.0001, and 0.002, respectively. e eGFR had a significant difference among the studied groups (p � 0.0001). Both urinary and plasma microRNA-451 showed a statistically significant difference in different patients' groups compared to the control group (p � 0.0001). Diabetic patients with macroalbuminuria showed the highest level of plasma microRNA-451 and the lowest level of both urinary microRNA-451 and eGFR (Table 1).
Comparison between the two groups showed no significant difference regarding age, sex, BUN, fasting blood sugar, HBA1c, total cholesterol, LDL, HDL, TGs, hemoglobin, WBCs, and platelets. On the other hand, there was a significant difference as regard creatinine, albuminuria, plasma microRNA-451, and urinary microRNA-451 with pvalues 0.0001, 0.035, 0.036, and 0.0001, respectively, as shown in Table 2. In the unshown data due to small sample size, subgroup analysis of patients with eGFR <60 ml/min/ 1.73 m 2 (five patients) showed a significant increase in plasma microRNA-451 and decrease in urinary microRNA-451 in comparison to the control group with p-values 0.01 and 0.008, respectively. e eGFR showed a significantly negative correlation with albuminuria and plasma microRNA-451, r � −0.491, p � 0.0001 and r � −0.458, p � 0.0001, respectively, and a positive correlation with urinary microRNA-451 (r � 0.262, p � 0.013) as shown in Table 3.
For the determination of sensitivity and specificity of microRNA-451 in blood and urine as biomarkers for early detection of diabetic nephropathy and chronic kidney disease in our patients, ROC analysis was used. e area under the curve was 0.427 for urinary microRNA-451 and 0.625 for plasma microRNA-451. e sensitivity of plasma micro-RNA-451 was 90.9% for cutoff 27.5 with a specificity of 67.7%, while the sensitivity of plasma microRNA-451 was 95.5% for cutoff 19.5 with specificity was 95.6%, as shown Table 4.

Discussion
Diabetes mellitus is the leading cause of end-stage renal disease worldwide [21]. Early detection of diabetic nephropathy is still a challenge. Regular followup urinary albumin is still the cornerstone for early evaluation of renal involvement in diabetic patients. However, there are patients with a decline in renal functions not accompanied by microalbuminuria or increased creatinine levels [22,23]. In the present study, 16.7% of patients with normoalbuminuria had reduced eGFR, which was in agreement with Krolewski et al., who stated that approximately 10-30% of diabetic patients develop renal impairment before the onset of microalbuminuria or macroalbuminuria [24]. e change in creatinine level, together with increased albumin in urine, appears as an attractive biomarker for the diagnosis of glomerular dysfunction associated with DM. However, these markers have many limitations, such as the tubular affection in DM, which may precede glomerular dysfunction leading to normal serum creatinine and normal level of urine albumin [25]. erefore, there is a need for new, easily accessible, and noninvasive biomarkers for early detection of diabetic nephropathy. Among these biomarkers is the microRNA expression, which are affected by the pathological conditions involving kidneys [26].

International Journal of Nephrology
In the present study, plasma microRNA-451 showed a significant increase in patients with DM and mild to moderate end-stage renal disease either with or without albuminuria. ese findings were similar to the previous experimental study by Zhang et al. [27,28]. e increased expression of miR-451 has a protective role in DN that leads to the inhibition of glomerular and mesangial cell proliferation, in both in vivo and in vitro studies.
is pathway is mediated through the downregulation of Ywhaz and p38 MAPK signal pathway. ese are the target genes for miR-451, which downregulated the expression of Ywhaz (tyrosine3-monooxygenase, tryptophan 5 monooxygenase activation protein, and zeta) gene in the 3′ UTR. e overexpression of miR-451 caused the   reduced activities of the two kinases (p38 MAPK and MKK3) and the suppression of Ywhaz [28]. erefore, this miRNA may be a potential target for intervention and prevention of DN in an early stage. On the other hand, urinary microRNA-451 in the present study showed a significant reduction in patients with eGFR <60 ml/min/1.73 m 2 compared to the other group of patients with eGFR >60 ml/min/1.73 m 2 . Our result matched the previous study which reported a reduction of all miRNA levels as they decreased in parallel with the renal functions among patients with end-stage renal disease [29]. e higher levels of microRNA-451 expression in plasma in association with its low expression in urine samples in patients with low eGFR were in agreement with previous studies in diabetic nephropathy patients [26] and hypertension associated with kidney affection [30]. ese findings indicate that the expression of microRNA-451 in blood and urine can be an accurate marker for early diagnosis of DN even in the absence of albuminuria.
ere was a significant positive correlation between the urinary microRNA-451 level and the eGFR value in association with a negative correlation between the plasma microRNA-451 level and eGFR, which supports the role of microRNA-451 as a predictor of disease progression and the development of renal fibrosis [30]. Previous studies for microRNA-451 revealed an early increase in its level in the tissue of the kidney and later on a decrease in urine, reflecting the increased activity of signal pathways such as MAPK9 and SMAD3 in association with the process of nephropathy development [27,30]. erefore, the expression of microRNA-451 in urine might reflect the glomerular filtration rate and renal production of this microRNA. e question raised either from this study and/or the previous studies was about the use of microRNA-451 as a predictor for the disease progression and a therapeutic approach to prevent the development of DN. is approach can be mediated through miRNA mimics, miRNA expression vectors, miRNA-containing exosomes, and miRNAinducing natural agents with the restoration of renal-protective miRNAs levels and subsequent protection from DN [31].
e present study has shown that both plasma and urinary microRNA-451 can be used as a sensitive and specific marker for the early diagnosis of DN. e sensitivity of plasma microRNA-451 was 90.9% with a specificity of 67.6%, while the sensitivity of urinary microRNA-451 was 95.5% with specificity 99.6%, which were in agreement with the results of Sayilar et al. [27]. On the other hand, binary logistic regression showed that plasma microRNA-451 was a significant independent predictor for eGFR <60 mL/min/ 1.73 m 2 compared to urinary microRNA-451, as shown in Table 5.

Conclusion
e present study's findings highlight the different expressions of microRNA-451 in blood and urine in different stages of diabetic nephropathy.
ere is a marked and gradual increase in blood level in different stages, denoting the potential role of this microRNA in the pathogenesis of this condition and its usefulness as an early biomarker for diagnosis. Moreover, the level of urinary microRNA-451 has a gradual reduction, reflecting the role played in the pathogenesis, and makes it a valuable biomarker for early diagnosis of diabetic nephropathy even in the absence of albuminuria. is study has its limitations, including a small sample size and the lack of renal biopsy and pathology, which we think will add a lot of value to our results.

Data Availability
All the data used to support the finding of this study are included in the manuscript and in the supplementary materials.