MiRNA-200b level in peripheral blood predicts renal interstitial injury in patients with diabetic nephropathy

Background To uncover the diagnostic potential of peripheral blood microRNA-200b (miRNA-200b) in renal interstitial injury in diabetic nephropathy (DN) patients. Methods A total of 50 diabetes subjects, 50 mild DN subjects, 50 moderate-severe DN subjects and 50 healthy subjects were included. Peripheral blood level of miRNA-200b in every subject was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Serum levels of renal function indicators were determined by enzyme-linked immunosorbent assay (ELISA). Meanwhile, relative levels of fibrosis damage indicators were examined by chemiluminescent immunoassay. Diagnostic potentials of miRNA200b in diabetes, mild DN and moderate-severe DN were assessed by depicting receiver operating characteristic (ROC) curves. Results Peripheral blood level of miRNA-200b was higher in DN subjects than diabetes subjects without vascular complications, especially moderate-severe DN patients. Peripheral blood level of miRNA-200b in DN subjects was negatively correlated to relative levels of serum creatinine, urinary nitrogen, cystatin, TGF-b, CIV and PCIII. ROC curves demonstrated diagnostic potentials of miRNA-200b in mild and moderate-severe DN. Conclusions Peripheral blood level of miRNA-200b is closely linked to the degree of renal interstitial injury in DN patients. MiRNA-200b may be a vital indicator in predicting the development of DN.


Summary
Background: To uncover the diagnostic potential of peripheral blood microRNA-200b (miRNA-200b) in renal interstitial injury in diabetic nephropathy (DN) patients. Methods: A total of 50 diabetes subjects, 50 mild DN subjects, 50 moderate-severe DN subjects and 50 healthy subjects were included. Peripheral blood level of miRNA-200b in every subject was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Serum levels of renal function indicators were determined by enzyme-linked immunosorbent assay (ELISA). Meanwhile, relative levels of fibrosis damage indicators were examined by chemiluminescent immunoassay. Diagnostic potentials of miRNA-200b in diabetes, mild DN and moderate-severe DN were assessed by depicting receiver operating characteristic (ROC) curves. Results: Peripheral blood level of miRNA-200b was higher in DN subjects than diabetes subjects without vascular complications, especially moderate-severe DN patients. Peripheral blood level of miRNA-200b in DN subjects was negatively correlated to relative levels of serum creatinine, urinary nitrogen, cystatin, TGF-b, CIV

Introduction
Diabetic nephropathy (DN) is an important microvascular complication of diabetes, which is the most common cause of end-stage renal failure (ESRD) (1). It is estimated that by 2045, the number of diabetes patients worldwide will reach 693 million (2). Sustained hyperglycemia results in extensive vascular damage to eyes, kidneys, heart, and nerves. About 40% of diabetic patients are susceptible to DN (3). At present, renal biopsy and urine microalbumin detection are the major approaches to diagnose and monitor DN. However, renal biopsy is an invasive examination that is not acceptable to every DN patients and it fails to reflect the severity of DN (4). It is of significance to develop effective and specific biomarkers of DN.
MicroRNAs (miRNAs) are endogenous, singlestranded RNAs containing 21-25 nucleotides (5). They are tissue-and time-specific. Through inducing mRNA degradation and blocking protein translation, miRNAs exert post-transcriptional regulations (6). Functionally, miRNAs are extensively involved in early embryonic development, gene expressions, cell phenotypes, etc. (7,8). They also display a certain role in the development of kidney diseases (9). Detection of miRNA levels in blood or urine may contribute to early screening and disease monitoring of DN.
MiRNA-200 family is a cluster of epithelial-mesenchymal transition (EMT)-associated miRNAs. In particular, miRNA-200b is considered as a negative regulator in tumor metastasis (10). It is reported that miRNA-200b initiates EMT by interacting with ZEB1/2 (11). Intercellular TAMs actively participate in tumor neovascularization by regulating EMT and enhancing tumor microvessel density (12). A relevant study showed that miRNA-200b protects diabetic retinopathy by downregulating VEGFA (13). Our study aims to uncover the role of miRNA-200b in the development of DN and its diagnostic potential.

Baseline characteristics
This study was performed after obtaining the approval of The Ethic Committee of Shanghai Sixth People's Hospital and the informed consent from the subjects. A total of 50 diabetes subjects without any vascular complications, 50 mild DN subjects (Mogensen II) and 50 moderate-severe DN subjects (Mogensen III-IV) were included. During the same period, 50 healthy subjects undergoing healthy examinations were included. Diabetes and DN were diagnosed based on the standard criteria (14) and kidney biopsy, respectively. Inclusion criteria were: (1) Diagnosis as type 2 diabetes mellitus; (2)  Blood sample collection 5 mL of venous blood was extracted in each subject under the fasting state in the morning. Blood was centrifuged at 3,000 r/min for 10 min, and the serum was collected and stored at -80 °C.

Determination of serum markers
Renal function indicators, including serum creatinine, urinary nitrogen, uric acid, and cystatin C were measured through enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA) by sarcosine oxidase method, immunoturbidimetry method, enzymatic method and immunoturbidimetry, respectively. Fibrosis damage indicators were examined by chemiluminescent immunoassay.

Statistical analyses
Statistical Product and Service Solutions (SPSS) 20.0 (IBM, Armonk, NY, USA) was used for all statistical analysis. Data were expressed as mean ± SD (standard deviation). Differences between two groups were analyzed by using the Student's t-test. Comparison between multiple groups was done using One-way ANOVA test followed by Post Hoc Test (Least Significant Difference). Pearson correlation test was conducted for assessing the relationship between miRNA-200b level and serum markers. Receiver operating characteristic (ROC) curves were depicted for evaluating diagnosis potentials of miRNA-200b. P<0.05 indicated the significant difference.  (Table I).

Peripheral blood level of miRNA-200b
RT-PCR data showed that peripheral blood level of miRNA-200b was higher in healthy subjects than diabetes and DN subjects. In particular, miRNA-200b level was lower in DN subjects than diabetes subjects, especially moderate-severe DN subjects (Table II). It is indicated that miRNA-200b may be favorable to prevent DN development.

Renal function indicators
Relative levels of serum creatinine, urinary nitrogen, uric acid and cystatin were lower in healthy subjects than diabetes and DN subjects. Notably, the highest levels of renal function indicators were found in moderate-severe DN subjects, followed by mild DN subjects and diabetes subjects (Table III). We believed that renal function indicators contribute to assess the severity of DN.

Serum markers of fibrosis damage
Serum markers of fibrosis damage, including TGF-b, HA, CIV and PCIII were examined in each subject. Relative levels of fibrosis damage indicators were lower in healthy subjects than diabetes and DN subjects. The highest levels were seen in moderatesevere DN subjects (Table IV). Therefore, serum markers of fibrosis damage may also be used to assess the severity of DN.

Pearson correlation test on miRNA-200b level and serum markers
We have proven that relative levels of miRNA-200b, renal function indicators, and serum markers of fibrosis damage were different in diabetes and DN subjects. Subsequently, Pearson correlation test showed that peripheral blood level of miRNA-200b was negatively correlated to serum creatinine, urinary nitrogen, cystatin, TGF-b, CIV and PCIII (r = -0.521, -0.683, -0.683, -0.811, -0.588 and -0.721, respectively) in DN subjects ( Table V).

Discussion
It is estimated that by 2030, 7.7% of people aging 20-79 years suffer from diabetes (16). DN is a severe complication of diabetes. Uncontrolled DN will deteriorate into ESRD that is difficult to be treated. Current therapeutic strategies of DN aim to control blood glucose, blood pressure and lipids (17). Nevertheless, the development of DN cannot be reversed or blocked. Prevention and intervention of DN in the early stage are of significance.
A single miRNA can bind several target genes, thereafter influencing gene expressions and functions (18). Differentially expressed miRNAs in kidney tissues of DN patients are able to reflect the disease condition (19). MiRNAs are stably expressed in serum, and detection of serum miRNAs is sensitive and specific (20). It is reported that miRNAs are involved in thickening of the glomerular basement membrane, podocyte apoptosis, deposition of extracellular matrix, cell fibrosis, etc., and eventually lead to the development of DN (21). MiRNAs are believed as promising biomarkers in diagnosis and monitoring of DN. Bai et al. (22) proposed that miRNA-130b is downregulated in kidney tissues of DN patients. MiRNA-130b alleviates EMT-induced fibrosis in rat renal tubular epithelial cells through downregulating Snail. A prospective study conducted in Europe involving 455 type 1 diabetes mellitus patients uncovered that serum level of miRNA-126 is negatively linked to susceptibilities to diabetic vascular complications, especially proliferative kidney diseases (23).
In this trial, we found out that miRNA-200b level was downregulated in peripheral blood of DN subjects, especially moderate-severe DN subjects. Subsequently, potential relationship between miRNA-200b level and renal function and fibrosis damage indicators was analyzed. Pearson correlation test showed that peripheral blood level of miRNA-200b was negatively correlated to serum creatinine, urinary nitrogen, cystatin, TGF-b, CIV and PCIII in DN patients. Such a correlation was not identified in diabetes patients without vascular complications, suggesting that renal function may be normal in diabetes patients. ROC curves analyses further demonstrated the diagnostic potentials of miRNA-200b in mild and moderate-severe DN. However, there are still two shortcomings in this study. Firstly, evaluation indicators of renal interstitial fibrosis lack organ specificity. Secondly, the role of miRNA-200b may be varied in DN with different pathological stages. Our results should be validated in future explorations.
Several previous studies demonstrated that miR-200 family may be involved in the development of diabetic nephropathy, but most of these studies focused on molecular mechanisms rather than directly analyzing clinical data through peripheral blood samples of patients (24,25). Compared with previous studies, the most significant innovation of this study is that it is the first study to focus on the expression level of miR-200b in peripheral blood of patients with diabetic nephropathy and its clinical value.

Conclusions
Peripheral blood level of miRNA-200b is closely linked to the degree of renal interstitial injury in DN patients. MiRNA-200b may be a vital indicator in predicting the development of DN.

Financial Disclosure
The authors declared that this study has received no financial support.